Tau antisense oligomers and uses thereof

ABSTRACT

The present invention relates to oligomer compounds (oligomers), which target Tau mRNA in a cell, leading to reduced expression of Tau protein. Reduction of Tau protein expression is beneficial for the treatment of certain medical disorders, e.g., a neurological disorder.

REFERENCE TO EARLIER FILED APPLICATIONS

This application is a national stage entry of PCT/US2016/016646, filedFeb. 4, 2016, which claims priority to U.S. Provisional Application No.62/112,058, filed Feb. 4, 2015, U.S. Provisional Application No.62/156,684, filed May 4, 2015, U.S. Provisional Application No.62/237,922, filed Oct. 6, 2015, U.S. Provisional Application No.62/238,941, filed Oct. 8, 2015, U.S. Provisional Application No.62/279,612, filed Jan. 15, 2016, U.S. Provisional Application No.62/279,614, filed Jan. 15, 2016, and U.S. Provisional Application No.62/279,610, filed Jan. 15, 2016, each of which is incorporated herein byreference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing named“2019-08-22 Sequence-Listing-Amended.txt” which was created on Aug. 22,2019 and is 380,928 bytes in size submitted in this application isincorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to oligomeric compounds (oligomers) thattarget microtubule-associated protein tau (MAPT) transcript in a cell,leading to reduced expression of MAPT mRNA and/or Tau protein. Reductionof MAPT mRNA and/or Tau protein expression is beneficial for a range ofmedical disorders, such as tauopathies, Down syndrome, depression,seizure disorders, and movement disorders.

BACKGROUND

Tau protein is a microtubule-associated protein (MAP) that interactswith tubulin to stabilize, and promote assembly into, microtubules.Microtubules are critical structural components of the cellularcytoskeleton and are involved in various cellular processes, includingmitosis, cytokinesis, and vesicular transport. Tau protein is present inmultiple cell and tissue types, but is particularly abundant in neuronscompared to non-neuronal cells.

Due to Tau's role in stabilizing microtubules, alteration of Tauexpression levels and/or function can disrupt critical cellularprocesses, which is thought to contribute to various neurodegenerativedisorders such as tauopathies. For example, it has been found thatneurofibrillary inclusions in Alzheimer's disease (AD) containaggregates of hyperphosphorylated Tau protein.

In addition, abnormal Tau expression and/or function has been associatedwith other diseases of the brain (also included in the family ofpathologically and genetically defined tauopathies), includingFrontotemporal dementia (FTD), progressive supranuclear palsy (PSP),corticobasal degeneration (CBD), corticobasal ganglionic degeneration,dementia pugilistica, Frontotemporal dementia with parkinsonism linkedto chromosome 17 (FTDP-17), Lytico-Bodig disease, tangle-predominantdementia, ganglioglioma, gangliocytoma, meningioangiomatosis, subacutesclerosing panencephalitis, lead encephalopathy, tuberous sclerosis,Hallervorden-Spatz disease, Pick's disease, argyrophilic grain disease,corticobasal degeneration or frontotemporal lobar degeneration, andothers. Abnormal Tau expression and/or function can also play a role inadditional diseases such as Down Syndrome, seizure disorders (e.g.,epilepsy), network dysfunction (e.g., depression), and movementdisorders (e.g., Parkinson's disease).

Tau-associated disorders such as AD are the most common cause ofdementia in the elderly, and robust and effective agents for thetreatment of neurodegenerative diseases, including tauopathies, seizuredisorders, and movement disorders, are greatly needed.

Antisense molecules that can decrease protein expression have beenstudied in the development of human therapeutics. Antisense moleculesthat target pre-mRNA or mRNA can reduce the RNA level thereby reducingthe protein level. Antisense molecules can act on a target sequencethrough various mechanisms of action: degradation of mRNA throughRNaseH, steric hindrance of ribosomal subunit binding, alteringmaturation of mRNA, splicing activation, 5′-cap formation inhibition,arrest of translation and/or double strand RNase activation. In somecases, however, antisense molecules targeting regions nearbypolyadenylation sites are known to increase mRNA stability. See Vickerset al., NAR (2001) 29(6) 1293-1299.

SUMMARY OF INVENTION

The present invention provides an oligomer of from 10 to 50 nucleotidesin length comprising a contiguous nucleotide sequence that hybridizes toa nucleic acid sequence within a microtubule-associated protein tau(MAPT) transcript, wherein the nucleic acid sequence corresponds tonucleotides 134,947-138,940 of SEQ ID NO: 1.

The present invention also provides an oligomer of from 10 to 50nucleotides in length comprising a contiguous nucleotide sequence thathybridizes to a nucleic acid sequence within a microtubule-associatedprotein tau (MAPT) transcript, wherein the nucleic acid sequencecorresponds to nucleotides 135,050-138,940 of SEQ ID NO: 1.

The present invention further provides an oligomer of from 10 to 50nucleotides in length comprising a contiguous nucleotide sequence thathybridizes to a nucleic acid sequence within a microtubule-associatedprotein tau (MAPT) transcript, wherein the nucleic acid sequencecorresponds to nucleotides 72,802-73,072 of SEQ ID NO: 1.

The present invention also provides an oligomer of from 10 to 50nucleotides in length comprising a contiguous nucleotide sequence thathybridizes to a nucleic acid sequence within a microtubule-associatedprotein tau (MAPT) transcript, wherein the oligomer has at least oneproperty selected from: (1) reduces expression of Tau mRNA in a cell,compared to a control cell that has not been exposed to the oligomer;and (2) reduces expression of Tau protein in a cell, compared to acontrol cell that has not been exposed to the oligomer.

The present invention also provides an oligomer of from 10 to 50nucleotides in length comprising a contiguous nucleotide sequence thathybridizes to a nucleic acid sequence within a microtubule-associatedprotein tau (MAPT) transcript, wherein the oligomer has an in vivotolerability less than or equal to a total score of 4, wherein the totalscore is the sum of a unit score of five categories, which are 1)hyperactivity; 2) decreased activity and arousal; 3) motor dysfunctionand/or ataxia; 4) abnormal posture and breathing; and 5) tremor and/orconvulsions, and wherein the unit score for each category is measured ona scale of 0-4.

The present invention also provides a conjugate comprising an oligomerof from 10 to 50 nucleotides in length comprising a contiguousnucleotide sequence that hybridizes to a nucleic acid sequence within amicrotubule-associated protein tau (MAPT) transcript, wherein theoligomer is covalently attached to at least one non-nucleotide ornon-polynucleotide moiety.

The present invention also provides a pharmaceutical compositioncomprising an oligomer of from 10 to 50 nucleotides in length comprisinga contiguous nucleotide sequence that hybridizes to a nucleic acidsequence within a microtubule-associated protein tau (MAPT) transcriptand a pharmaceutically acceptable carrier.

The present invention also provides a kit comprising an oligomer of from10 to 50 nucleotides in length comprising a contiguous nucleotidesequence that hybridizes to a nucleic acid sequence within amicrotubule-associated protein tau (MAPT) transcript and instructionsfor use.

The present invention further provides a method of inhibiting orreducing Tau protein expression in a cell, the method comprisingadministering an oligomer of from 10 to 50 nucleotides in lengthcomprising a contiguous nucleotide sequence that hybridizes to a nucleicacid sequence within a microtubule-associated protein tau (MAPT)transcript to a cell expressing Tau protein, wherein the Tau proteinexpression in the cell is inhibited or reduced after the administration.

The present invention further provides a method for treating a seizuredisorder in a subject in need thereof, the method comprisingadministering an oligomer of from 10 to 50 nucleotides in lengthcomprising a contiguous nucleotide sequence that hybridizes to a nucleicacid sequence within a microtubule-associated protein tau (MAPT)transcript to a cell expressing Tau protein to the subject.

The present invention further provides a method for treating a seizuredisorder in a subject in need thereof, the method comprisingadministering an oligomer of from 10 to 50 nucleotides in lengthcomprising a contiguous nucleotide sequence that hybridizes to a nucleicacid sequence within a microtubule-associated protein tau (MAPT)transcript to a cell expressing Tau protein to the subject.

The present invention further provides the use of an oligomer of from 10to 50 nucleotides in length comprising a contiguous nucleotide sequencethat hybridizes to a nucleic acid sequence within amicrotubule-associated protein tau (MAPT) transcript for the manufactureof a medicament for the treatment of a neurological disorder, e.g., atauopathy, a neurodegenerative disease with tauopathy (aneurodegenerative disease which involves accumulation of tau protein inthe brain), an epileptic disorder with tauopathy (an epileptic disorderwhich involves accumulation of tau protein in the brain), an epilepticdisorder without tauopathy (an epileptic disorder which does not involveaccumulation of tau protein in the brain), an idiopathic adult epilepticdisorder without tauopathy (an idiopathic adult epileptic disorder whichdoes not involve accumulation of tau protein in the brain), a seizuredisorder, or any combination thereof.

Embodiments

E1. An oligomer of from 10 to 50 nucleotides in length comprising acontiguous nucleotide sequence that hybridizes to a nucleic acidsequence within a microtubule-associated protein tau (MAPT) transcript,wherein the nucleic acid sequence corresponds to nucleotides134,947-138,940 of SEQ ID NO: 1.

E2. An oligomer of from 10 to 50 nucleotides in length comprising acontiguous nucleotide sequence that hybridizes to a nucleic acidsequence within a microtubule-associated protein tau (MAPT) transcript,wherein the nucleic acid sequence corresponds to nucleotides135,050-138,940 of SEQ ID NO: 1.

E3. An oligomer of from 10 to 50 nucleotides in length comprising acontiguous nucleotide sequence that hybridizes to a nucleic acidsequence within a microtubule-associated protein tau (MAPT) transcript,wherein the nucleic acid sequence corresponds to nucleotides72,802-73,072 of SEQ ID NO: 1.

E4. The oligomer of any one of embodiments 1 or 3, wherein thenucleotide sequence comprises at least one nucleotide analog.

E5. The oligomer of any one of embodiments 1 to 4, which is a gapmer, ablockmer, a mixmer, a headmer, a tailmer, or a totalmer.

E6. The oligomer of any one of embodiments 1 to 5, which is a gapmer.

E7. The oligomer of embodiment 6, which has the formula of 5′-A-B-C-3′(II), wherein

-   (i) B is a contiguous sequence of 7 to 23 DNA units;-   (ii) A is a first wing sequence of 1 to 10 nucleotides, wherein the    first wing sequence comprises one or more nucleotide analogs and    optionally one or more DNA units and wherein at least one of the    nucleotide analogs is located at the 5′ end of A; and-   (iii) C is a second wing sequence of 1 to 10 nucleotides, wherein    the second wing sequence comprises one or more nucleotide analogs    and optionally one or more DNA units and wherein at least one of the    nucleotide analogs is located at the 3′ end of C.

E8. The oligomer of embodiment 7, wherein A has the fomula of LmDnLoDpLq(III) and C has the fomula of Lm′Dn′Lo′Dp′Lq′ (IV) and wherein

-   L is a nucleotide analog;-   D is a DNA unit;-   m and q′ are 1 to 6 units;-   n, p, n′, and p′ are 0 to 2 units; and-   o, q, m′, and o′ are 0 to 5 units.

E9. The oligomer of embodiment 7 or 8, wherein the first wing sequencecomprises a combination of nucleotide analogs and DNA unit selected from(i) 1-9 nucleotide analogs and 1 DNA unit; (ii) 1-8 nucleotide analogsand 1-2 DNA units; (iii) 1-7 nucleotide analogs and 1-3 DNA units; (iv)1-6 nucleotide analogs and 1-4 DNA units; (v) 1-5 nucleotide analogs and1-5 DNA units; (vi) 1-4 nucleotide analogs and 1-6 DNA units; (vii) 1-3nucleotide analogs and 1-7 DNA units; (viii) 1-2 nucleotide analogs and1-8 DNA units; and (ix) 1 nucleotide analog and 1-9 DNA units.

E10. The oligomer of any one of embodiments 7 to 9, wherein the secondwing sequence comprises a combination of nucleotide analogs and DNA unitselected from (i) 1-9 nucleotide analogs and 1 DNA unit; (ii) 1-8nucleotide analogs and 1-2 DNA units; (iii) 1-7 nucleotide analogs and1-3 DNA units; (iv) 1-6 nucleotide analogs and 1-4 DNA units; (v) 1-5nucleotide analogs and 1-5 DNA units; (vi) 1-4 nucleotide analogs and1-6 DNA units; (vii) 1-3 nucleotide analogs and 1-7 DNA units; (viii)1-2 nucleotide analogs and 1-8 DNA units; and (ix) 1 nucleotide analogand 1-9 DNA units.

E11. The oligomer of any one of embodiments 8 to 10, wherein A isselected from L, LL, LDL, LLL, LLLL, LLDL, LDLL, LDDL, LLDD, LLLLL,LLLDL, LLDLL, LDLLL, LLDDL, LDDLL, LLDLD, LDLLD, LDLDL, LDDDL, LLLLLL,LLLLDL, LLLDLL, LLDLLL, LDLLLL, LLLDDL, LLDLDL, LLDDLL, LDDLLL, LDLLDL,LDLDLL, LDDDLL, LLDDDL, and LDLDLD, and C is selected from L, LL, LDL,LLL, LLLL, LLDL, LDLL, LDDL, LLDD, LLLLL, LLLDL, LLDLL, LDLLL, LLDDL,LDDLL, LLDLD, LDLLD, LDLDL, LDDDL, LLLLLL, LLLLDL, LLLDLL, LLDLLL,LDLLLL, LLLDDL, LLDLDL, LLDDLL, LDDLLL, LDLLDL, LDLDLL, LDDDLL, LLDDDL,and LDLDLD.

E12. The oligomer of any one of embodiments 1 to 11, which comprises atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or at least tennucleotide analogs.

E13. The oligomer of any one of embodiments 1 to 12, wherein thenucleotide analog or analogs are selected from Locked Nucleic Acid(LNA); 2′-O-alkyl-RNA; 2′-amino-DNA; 2′-fluoro-DNA; arabino nucleic acid(ANA); 2′-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleicacid (INA), constrained ethyl nucleoside (cEt), 2′-O-methyl nucleic acid(2′-OMe), 2′-O-methoxyethyl nucleic acid (2′-MOE), and any combinationthereof.

E14. The oligomer of any one of embodiments 1 to 13, wherein thenucleotide analog or analogs comprise a bicyclic sugar.

E15. The oligomer of embodiment 14, wherein the bicyclic sugar comprisescEt, 2′,4′-constrained 2′-O-methoxyethyl (cMOE), LNA, α-LNA, β-LNA,2′-O,4′-C-ethylene-bridged nucleic acids (ENA), amino-LNA, oxy-LNA, orthio-LNA.

E16. The oligomer of any one of embodiments 1 to 15, wherein thenucleotide analog or analogs comprise an LNA.

E17. The oligomer of embodiment 16, which comprises three to five LNAson the 5′ portion of the oligomer and three to five LNAs on the 3′portion of the oligomer.

E18, The oligomer of any one of embodiments 1 and 4-17, wherein thenucleic acid sequence corresponds to nucleotides 134,947-138,924 of SEQID NO: 1.

E19. The oligomer of any one of embodiments 1 to 18, wherein the MAPTtranscript comprises SEQ ID NO: 1.

E20. The oligomer of embodiment 19, wherein the nucleotide sequencehybridizes to a nucleic acid sequence within nucleotides135,700-138,940; 136,000-138,940; 136,620-138,940; 136,860-138,940;137,060-138,940; 137,300-138,940; 137,830-138,940; 138,030-138,940;138,350-138,940; 134,821-135,020; 135,700-135,820; 136,000-136,110;136,620-136,760; 136,860-136,960; 137,060-137,110; 137,300-137,400;137,830-137,900; 138,030-138,140; 138,350-138,450; or 138,860-138,940 ofSEQ ID NO: 1.

E21. The oligomer of embodiment 19, wherein the nucleotide sequence hasat least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% sequence identity to a region within the complement of a nucleicacid sequence selected from nucleotides 134,947-138,940 of SEQ ID NO: 1.

E22. The oligomer of embodiment 21, wherein the nucleotide sequence hasat least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% sequence identity to a region within the complement ofnucleotides 135,700-138,940; 136,000-138,940; 136,620-138,940;136,860-138,940; 137,060-138,940; 137,300-138,940; 137,830-138,940;138,030-138,940; 138,350-138,940; 134,821-135,020; 135,700-135,820;136,000-136,110; 136,620-136,760; 136,860-136,960; 137,060-137,110;137,300-137,400; 137,830-137,900; 138,030-138,140; 138,350-138,450; or138,860-138,940 of SEQ ID NO: 1.

E23. The oligomer of any one of embodiments 3 to 17, wherein the nucleicacid sequence corresponds to nucleotides 72,802-73,072; 72,812-73,062;72,822-73,052; 72,832-73,042; 72,842-73,032; 72,852-73,022;72,862-73,012; 72,872-73,002; 72,882-72,992; 72,892-72,982; or72,902-72,972 of SEQ ID NO: 1.

E24. The oligomer of embodiment 22, wherein the nucleotide sequencehybridizes to a nucleic acid sequence within nucleotides 72,802-73,072;72,812-73,062; 72,822-73,052; 72,832-73,042; 72,842-73,032;72,852-73,022; 72,862-73,012; 72,872-73,002; 72,882-72,992;72,892-72,982; or 72,902-72,972 of SEQ ID NO: 1.

E25. The oligomer of embodiment 23, wherein the nucleotide sequence hasat least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% sequence identity to a region within the complement ofnucleotides 72,862-73,012; 72,872-73,012; 72,882-73,012; 72,892-73,012;72,902-73,012; 72,862-73,002; 72,872-73,002; 72,882-73,002;72,892-73,002; 72,902-73,002; 72,862-72,992; 72,872-72,992;72,882-72,992; 72,892-72,992; 72,902-72,992; 72,862-72,982;72,872-72,982; 72,882-72,982; 72,892-72,982; 72,902-72,982;72,862-72,972; 72,872-72,972; 72,882-72,972; 72,892-72,972; or72,902-72,972 of SEQ ID NO: 1.

E26. The oligomer of embodiment 24, wherein the nucleotide sequence hasat least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% sequence identity to a region within the complement ofnucleotides 72,862-73,012; 72,872-73,012; 72,882-73,012; 72,892-73,012;72,902-73,012; 72,862-73,002; 72,872-73,002; 72,882-73,002;72,892-73,002; 72,902-73,002; 72,862-72,992; 72,872-72,992;72,882-72,992; 72,892-72,992; 72,902-72,992; 72,862-72,982;72,872-72,982; 72,882-72,982; 72,892-72,982; 72,902-72,982;72,862-72,972; 72,872-72,972; 72,882-72,972; 72,892-72,972; or72,902-72,972 of SEQ ID NO: 1.

E27. The oligomer of embodiment 24, wherein the nucleotide sequence hasat least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% sequence identity to a region within the complement ofnucleotides 72,947-72,960; 72,946-72,961; 72,907-72,922; 72,948-72,963;72,950-72,963; 72,945-72,960; 72,950-72,965; 72,944-72,959;72,947-72,962; 72,952-72,965; 72,946-72,959; 72,949-72,964;72,951-72,964; 72,933-72,948; 72,934-72,949; 72,935-72,950;72,932-72,951; 72,933-72,952; 72,934-72,953; 72,945-72,964;72,944-72,963; 72,948-72,967; 72,946-72,965; 72,935-72,951;72,936-72,953; 72,933-72,934; 72,933-72,954; 72,933-72,950;72,935-72,954; 72,934-72,951; 72,934-72,950; 72,933-72,949; or72,935-72,952 of SEQ ID NO: 1.

E28. The oligomer of any one of embodiments 1 to 27, wherein thenucleotide sequence comprises no mismatches or no more than one or twomismatches with the region.

E29. The oligomer of any one of embodiments 1, 2, 4-22, and 28, whereinthe nucleotide sequence has at least about 80%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% sequence identity to a region withinthe complement of nucleotides 136,000-136,110 or 138,860-138,940 of SEQID NO: 1.

E30. The oligomer of embodiment 29, wherein the nucleotide sequence hasat least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% sequence identity to a region within the complement ofnucleotides 136,053-136,068 or 138,884-138,908 of SEQ ID NO: 1.

E31. The oligomer of any one of embodiments 1, 2, 4-22, and 28, whereinthe nucleotide sequence comprises a nucleotide sequence at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a nucleic acid sequence selected from: SEQ ID NO: 4to SEQ ID NO: 803.

E32. The oligomer of any one of embodiments 1, 2, 4-22, and 28, whereinthe nucleotide sequence has at least about 80%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% sequence identity to a nucleic acidsequence selected from the sequences in FIGS. 2, 3, 6, and 7, whereinthe upper case letter is LNA and the lower case letter is DNA.

E33. The oligomer of any one of embodiments 1 to 17, wherein thenucleotide sequence has at least about 80%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or 100% sequence identity to a nucleic acid sequenceselected from: ctttatttccaaattcactt (SEQ ID NO: 676);actttatttccaaattcact (SEQ ID NO: 715); tttatttccaaattcacttt (SEQ ID NO:644); ttatttccaaattcactttt (SEQ ID NO: 799); atttccaaattcacttttac (SEQID NO: 466); atttccaaattcactttta (SEQ ID NO: 559); actttatttccaaattcactt(SEQ ID NO: 680); or atttccaaattcactt (SEQ ID NO: 686).

E34. The oligomer of embodiment 32, wherein the nucleotide sequence hasat least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% sequence identity to a nucleic acid sequence selected from:tatttccaaattcactttta (SEQ ID NO: 526); aataactttatttcca (SEQ ID NO:773); agtaataactttatt (SEQ ID NO: 782); tttccaaattcactt (SEQ ID NO:684); agagtaataactttat (SEQ ID NO: 784); agtaataactttattt (SEQ ID NO:780); agagtaataacttta (SEQ ID NO: 786); ttaatcagagtaataa (SEQ ID NO:795); tttaatcagagtaat (SEQ ID NO: 798); aatcagagtaataac (SEQ ID NO:794); tttaatcagagtaata (SEQ ID NO: 797); taatcagagtaataa (SEQ ID NO:796); ctttatttccaaattcact (SEQ ID NO: 713); and ctttatttccaaattcac (SEQID NO: 739).

E35. The oligomer of embodiment 32, wherein the nucleotide sequence hasat least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% sequence identity to a nucleic acid sequence selected from:atttccaaattcacttttac (SEQ ID NOs: 466 to 490, 513 to 525, 910 to 918,928, 929, or 932 to 935); tatttccaaattcactttta (SEQ ID NOs: 526 to 550,or 573 to 585); ttatttccaaattcactttt (SEQ ID NOs: 586-606, 629 to642,799 to 801); tttatttccaaattcacttt (SEQ ID NOs: 644 to 647, 657 to658, 919 to 921, or 930); ctttatttccaaattcactt (SEQ ID NOs: 676 to 679,681 to 683, 685, or 687 to 697); actttatttccaaattcactt (SEQ ID NO: 680);tttccaaattcactt (SEQ ID NO: 684); atttccaaattcactt (SEQ ID NOs: 686,705, 712, 936, or 937, 922, 924, or 931); actttatttccaaattcact (SEQ IDNOs: 715 to 717); ctttatttccaaattcact (SEQ ID NOs: 713, 714 or 718 to731,); ctttatttccaaattcac (SEQ ID NOs: 739 to 748); aataactttatttcca(SEQ ID NO: 773 or 774); agtaataactttattt (SEQ ID NO: 780);agtaataactttatt (SEQ ID NO: 782); agagtaataactttat (SEQ ID NO: 784);agagtaataacttta (SEQ ID NO: 786); aatcagagtaataac (SEQ ID NO: 794);ttaatcagagtaataa (SEQ ID NO: 795); taatcagagtaataa (SEQ ID NO: 796);tttaatcagagtaata (SEQ ID NO: 797); and tttaatcagagtaat (SEQ ID NO: 798)

E36. The oligomer of any one of embodiments 3 to 17, wherein thenucleotide sequence has at least about 80%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or 100% sequence identity to a nucleic acid sequenceselected from the sequences in FIGS. 16A and 16B, wherein the upper caseletter is LNA and the lower case letter is DNA.

E37. The oligomer of any one of embodiments 3 to 17, wherein thenucleotide sequence has at least about 80%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or 100% sequence identity to a nucleic acid sequenceselected from: SEQ ID NO: 804 to SEQ ID NO: 892.

E38. The oligomer of any one of embodiments 1 to 37, which issingle-stranded.

E39. The oligomer of any one of embodiments 1 to 38, which has at leastone property selected from: (1) reduces expression of Tau mRNA in acell, compared to a control cell that has not been exposed to theoligomer; and (2) reduces expression of Tau protein in a cell, comparedto a control cell that has not been exposed to the oligomer.

E40. The oligomer of any one of embodiments 1 to 39, wherein theoligomer has an in vivo tolerability less than or equal to a total scoreof 4, wherein the total score is the sum of a unit score of fivecategories, which are 1) hyperactivity; 2) decreased activity andarousal; 3) motor dysfunction and/or ataxia; 4) abnormal posture andbreathing; and 5) tremor and/or convulsions, and wherein the unit scorefor each category is measured on a scale of 0-4.

E41. The oligomer of embodiment 40, wherein the in vivo tolerability isless than or equal to the total score of 3, the total score of 2, thetotal score of 1, or the total score of 0.

E42. The oligomer of any one of embodiments 1 to 41, wherein calciumoscillations of neuronal cells which are in contact with the oligomerare greater than or equal to 95%, greater than or equal to 90%, greaterthan or equal to 85%, greater than or equal to 80%, greater than orequal to 75%, or greater than or equal to 70% of oscillations inneuronal cells that are not in contact with the oligomer.

E43. The oligomer of any one of embodiments 1 to 42, which reducesexpression of Tau mRNA in a cell by at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or about 100%compared to a cell not exposed to the oligomer.

E44. The oligomer of any one of embodiments 1 to 43, which reducesexpression of Tau protein in a cell by at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or at least about 95%compared to a cell not exposed to the oligomer.

E45. The oligomer of any one embodiments 1 to 44, which comprises thenucleotides A, T, C, and G and at least one analog of the nucleotides A,T, C, and G, and has a sequence score greater than or equal to 0.2,wherein the sequence score is calculated by formula I:

$\begin{matrix}{\frac{{E\#\mspace{14mu}{of}\mspace{14mu} C\mspace{14mu}{nucleotides}\mspace{14mu}{and}\mspace{14mu}{analogs}\mspace{14mu}{thereof}} - {\#\mspace{14mu}{of}\mspace{14mu} G\mspace{14mu}{nucleotides}\mspace{14mu}{and}\mspace{14mu}{analogs}\mspace{14mu}{thereof}}}{{Total}\mspace{14mu}{nucleotide}\mspace{14mu}{length}}.} & (I)\end{matrix}$

E46. The oligomer of any one of embodiments 1 to 45, which has from 10to 24 nucleotides in length or from 14 to 21 nucleotides in length.

E47. The oligomer of any one of embodiments 1 to 46, which has 14, 15,16, 17, 20, or 21 nucleotides in length.

E48. The oligomer of embodiment 1, 2, 4-22, and 28, which comprises,consists essentially of, or consists of a nucleotide sequence selectedfrom FIGS. 2, 3, 6, and 7, wherein the upper case letter is LNA and thelower case letter is DNA.

E49. The oligomer of embodiment 48, which comprises, consistsessentially of, or consists of: ATTtCcaaattcacTtTtAC (SEQ ID NO: 487);CTTTAtttccaaattCACTT (SEQ ID NO: 677); ACTTTatttccaaattCACT (SEQ ID NO:715); TTTATttccaaattcACTTT (SEQ ID NO: 644); TTtATttccaaattcACtTT (SEQID NO: 645); TTaTTtccaaattcaCTtTT (SEQ ID NO: 593); ATTTccaaattcactTTTAC(SEQ ID NO: 474); ACTTTatttccaaattCACTT (SEQ ID NO: 680);ATTtccaaattcaCTT (SEQ ID NO: 686); TATTTccaaattcactTTTA (SEQ ID NO:532); AATaactttatttCCA (SEQ ID NO: 773); AGTaataactttATT (SEQ ID NO:782); TTTccaaattcaCTT (SEQ ID NO: 684); AGAgtaataacttTAT (SEQ ID NO:784); AGTaataactttaTTT(SEQ ID NO: 780); AGAgtaataactTTA (SEQ ID NO:786); TTAatcagagtaaTAA (SEQ ID NO: 795); TTTaatcagagtAAT (SEQ ID NO:798); AATcagagtaatAAC (SEQ ID NO: 794); TTTaatcagagtaATA (SEQ ID NO:797); TAAtcagagtaaTAA (SEQ ID NO: 796); CTTtatttccaaatTCACT (SEQ ID NO:720); ATtTCcaaattcactTTtAC (SEQ ID NO: 472); AtTTCcaaattcactTTtAC (SEQID NO: 473); ATTtCcaaattcacTtTtAC (SEQ ID NO: 487); orCTTtatttccaaatTcAC (SEQ ID NO: 745), wherein the upper case letter isLNA and the lower case letter is DNA.

E50. The oligomer of embodiment 48, which comprises, consistsessentially of, or consists of: ATtTCcaaattcactTTtAC (SEQ ID NO: 472);AtTTCcaaattcactTTtAC (SEQ ID NO: 473); ATTTccaaattcactTTTAC (SEQ ID NO:474); ATTTCcaaattcacttTTAC (SEQ ID NO: 482); ATTtCcaaattcacTtTtAC (SEQID NO: 487); ATtTCcaaattcactTTtAC (SEQ ID NO: 524), AtTTCcaaattcactTTtAC(SEQ ID NO: 493), TATTTccaaattcactTTTA (SEQ ID NO: 532);TTaTTtccaaattcaCTtTT (SEQ ID NO: 593); TTTATttccaaattcACTTT (SEQ ID NO:644); TTtATttccaaattcACtTT (SEQ ID NO: 645); TTTATttccaaattcaCTTT (SEQID NO: 646), TTTAtttccaaattcACTTT (SEQ ID NO: 647); CTTTAtttccaaattCACTT(SEQ ID NO: 677); CTTTAtttccaaattcACTT (SEQ ID NO: 679);ACTTTatttccaaattCACTT (SEQ ID NO: 680); CTTTatttccaaattCACTT (SEQ ID NO:681); CTtTAtttccaaattCAcTT (SEQ ID NO: 683); TTTccaaattcaCTT (SEQ ID NO:684); CtTTAtttccaaattCAcTT (SEQ ID NO: 685); ATTtccaaattcaCTT (SEQ IDNO: 686); CTTtatttccaaatTcACT (SEQ ID NO: 714); ACTTTatttccaaattCACT(SEQ ID NO: 715); ACTTtatttccaaatTCACT (SEQ ID NO: 716);CTTtatttccaaatTCACT (SEQ ID NO: 720); CTTtatttccaaatTCAC (SEQ ID NO:740); CTTtatttccaaatTcAC (SEQ ID NO: 745); AATaactttatttCCA (SEQ ID NO:773); AGTaataactttaTTT(SEQ ID NO: 780); AGTaataactttATT (SEQ ID NO:782); AGAgtaataacttTAT (SEQ ID NO: 784); AGAgtaataactTTA (SEQ ID NO:786); AATcagagtaatAAC (SEQ ID NO: 794); TTAatcagagtaaTAA (SEQ ID NO:795); TAAtcagagtaaTAA (SEQ ID NO: 796); TTTaatcagagtaATA (SEQ ID NO:797); or TTTaatcagagtAAT (SEQ ID NO: 798), wherein the upper case letteris LNA and the lower case letter is DNA.

E51. The oligomer of embodiment 48, which comprises, consistsessentially of, or consists of CTTTAtttccaaattcACTT (SEQ ID NO: 679,ASO-001928), ATTTCcaaattcacttTTAC (SEQ ID NO: 482, ASO-001962);CTTTatttccaaattCACTT (SEQ ID NO: 681, ASO-001921), TTtATttccaaattcACtTT(SEQ ID NO: 645, ASO-001967); TTTAtttccaaattcACTTT (SEQ ID NO: 647,ASO-001948), TTaTTtccaaattcaCTtTT (SEQ ID NO: 593, ASO-001941),ACTTtatttccaaatTCACT (SEQ ID NO: 716, ASO-001956), CTtTAtttccaaattCAcTT(SEQ ID NO: 683, ASO-001942), TTTATttccaaattcaCTTT (SEQ ID NO: 646,ASO-001955), ACTTTatttccaaattCACTT (SEQ ID NO: 680, ASO-001968);CtTTAtttccaaattCAcTT (SEQ ID NO: 685, ASO-001935), AtTTCcaaattcactTTtAC(SEQ ID NO: 473, ASO-001933), TTtATttccaaattcACtTT (SEQ ID NO: 645,ASO-001967), ATtTCcaaattcactTTtAC (SEQ ID NO: 492), ATTtCcaaattcacTtTtAC(SEQ ID NO: 487, ASO-002038), or AtTTCcaaattcactTTtAC (SEQ ID NO: 493),wherein the upper case letter is LNA and the lower case letter is DNA.

E52. The oligomer of embodiment 50, which comprises, consistsessentially of, or consists of ATtTCcaaattcactTTtAC (SEQ ID NO: 472,ASO-001940); AtTTCcaaattcactTTtAC (SEQ ID NO: 473, ASO-001933),ATTTccaaattcactTTTAC (SEQ ID NO: 474; ASO-001919); ATTTCcaaattcacttTTAC(SEQ ID NO: 482, ASO-001962); ATTtCcaaattcacTtTtAC (SEQ ID NO: 487,ASO-002038), ATtTCcaaattcactTTtAC (SEQ ID NO: 524, ASO-002263),AtTTCcaaattcactTTtAC (SEQ ID NO: 493, ASO-002439), TATTTccaaattcactTTTA(SEQ ID NO: 532, ASO-001954); TTaTTtccaaattcaCTtTT (SEQ ID NO: 593,ASO-001941), TTTATttccaaattcACTTT (SEQ ID NO: 644, ASO-000756);TTtATttccaaattcACtTT (SEQ ID NO: 645, ASO-001967); TTTATttccaaattcaCTTT(SEQ ID NO: 646, ASO-001955), TTTAtttccaaattcACTTT (SEQ ID NO: 647,ASO-001948), CTTTAtttccaaattCACTT (SEQ ID NO: 677, ASO-000757);CTTTAtttccaaattcACTT (SEQ ID NO: 679, ASO-001928), ACTTTatttccaaattCACTT(SEQ ID NO: 680, ASO-001968); CTTTatttccaaattCACTT (SEQ ID NO: 681,ASO-001921), CTtTAtttccaaattCAcTT (SEQ ID NO: 683, ASO-001942),TTTccaaattcaCTT (SEQ ID NO: 684, ASO-000128); CtTTAtttccaaattCAcTT (SEQID NO: 685, ASO-001935), ATTtccaaattcaCTT (SEQ ID NO: 686, ASO-000013);CTTtatttccaaatTcACT (SEQ ID NO: 714, ASO-002012); ACTTTatttccaaattCACT(SEQ ID NO: 715, ASO-001962); ACTTtatttccaaatTCACT (SEQ ID NO: 716,ASO-001956), CTTtatttccaaatTCACT (SEQ ID NO: 720, ASO-001995);CTTtatttccaaatTCAC (SEQ ID NO: 740, ASO-002007); CTTtatttccaaatTcAC (SEQID NO: 745, ASO-001997); AATaactttatttCCA (SEQ ID NO: 773, ASO-000118);AGTaataactttaTTT(SEQ ID NO: 780, ASO-000170); AGTaataactttATT (SEQ IDNO: 782, ASO-000125); AGAgtaataacttTAT (SEQ ID NO: 784, ASO-000134);AGAgtaataactTTA (SEQ ID NO: 786, ASO-000178); AATcagagtaatAAC (SEQ IDNO: 794, ASO-000307); TTAatcagagtaaTAA (SEQ ID NO: 795, ASO-000204);TAAtcagagtaaTAA (SEQ ID NO: 796, ASO-000330); TTTaatcagagtaATA (SEQ IDNO: 797, ASO-000326); and TTTaatcagagtAAT (SEQ ID NO: 798, ASO-000249).

E53. The oligomer of embodiment 1 to 48, which comprises, consistsessentially of, or consists of a nucleotide sequence selected from FIGS.16A and 16B.

E54. The oligomer of embodiment 53, which comprises, consistsessentially of, or consists of a nucleotide sequence selected from FIGS.16A and 16B, wherein the upper case letter is LNA and the lower caseletter is DNA.

E55. The oligomer of any one of embodiments 1 to 54, which comprises aninternucleoside linkage selected from: a phosphodiester linkage, aphosphotriester linkage, a methylphosphonate linkage, a phosphoramidatelinkage, a phosphorothioate linkage, and combinations thereof.

E56. The oligomer of any one of embodiments 1 to 55, wherein theoligomer comprises a nucleotide analog.

E57. The oligomer of embodiment 56, wherein the nucleotide analogcomprises 5′methyl cytosine.

E58. A conjugate comprising the oligomer of any one of embodiments 1 to57, wherein the oligomer is covalently attached to at least onenon-nucleotide or non-polynucleotide moiety.

E59. The conjugate of embodiment 58, wherein the non-nucleotide ornon-polynucleotide moiety comprises a protein, a fatty acid chain, asugar residue, a glycoprotein, a polymer, or any combinations thereof.

E60. A pharmaceutical composition comprising the oligomer of any oneembodiments 1 to 57 or the conjugate of embodiment 58 or 59 and apharmaceutically acceptable carrier.

E61 The composition of embodiment 60, which further comprises atherapeutic agent.

E62. The composition of embodiment 61, wherein the therapeutic agent isa Tau antagonist.

E63. The composition of embodiment 62, wherein the Tau antagonist is ananti-Tau antibody or fragment thereof.

E64. A kit comprising the oligomer of any one embodiments 1 to 57, theconjugate of embodiment 58 or 59, or the composition of any one ofembodiments 60 to 63 and instructions for use.

E65. A diagnostic kit comprising the oligomer of any one embodiments 1to 57, the conjugate of embodiment 58 or 59, or the composition of anyone of embodiments 60 to 63 and instructions for use.

E66. A method of inhibiting or reducing Tau protein expression in acell, the method comprising administering the oligomer of any oneembodiments 1 to 57, the conjugate of embodiment 58 or 59, or thecomposition of any one of embodiments 60 to 63 to the cell expressingTau protein, wherein the Tau protein expression in the cell is inhibitedor reduced after the administration.

E67. The method of embodiment 66 wherein the oligomer inhibits orreduces expression of Tau mRNA in the cell after the administration.

E68. The method of embodiment 66 or 67, wherein the expression of TaumRNA is reduced by at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, or about 100% after theadministration compared to a cell not exposed to the oligomer.

E69. The method of any one of embodiments 66 to 68, wherein the oligomerreduces expression of Tau protein in the cell after the administrationby at least about 60%, at least about 70%, at least about 80%, or atleast about 90% compared to a cell not exposed to the oligomer.

E70. The method of any one of embodiments 66 to 69, wherein the cell isa neuron.

E71. A method for treating a tauopathy in a subject in need thereof,comprising administering an effective amount of the oligomer of any oneembodiments 1 to 57, the conjugate of embodiment 58 or 59, or thecomposition of any one of embodiments 60 to 63 to the subject.

E72. The method of embodiment 71, wherein the tauopathy is a diseaseselected from Alzheimer's disease, progressive supranuclear palsy,dementia pugilistica (chronic traumatic encephalopathy), frontaltemporal dementia, parkinsonism linked to chromosome 17, Lytico-Bodigdisease (Parkinson-dementia complex of Guam), Tangle-predominantdementia, ganglioglioma, gangliocytoma, meningioangiomatosis, subacutesclerosing panencephalitis, lead encephalopathy, tuberous sclerosis,Hallervorden-Spatz disease, Pick's disease, corticobasal ganglionicdegeneration, argyrophilic grain disease, corticobasal degeneration,lipofuscinosis, frontotemporal dementia, supranuclear palsy,frontotemporal lobar degeneration, and any combination thereof.

E73. The method of embodiment 71, wherein the tauopathy is progressivesupranuclear palsy.

E74. The method of embodiment 71, wherein the tauopathy is Alzheimer'sdisease.

E75. The method of embodiment 71, wherein the tauopathy is frontaltemporal dementia.

E76. A method of regulating neuronal hyperexcitability in a subject inneed thereof comprising administering an effective amount of theoligomer of any one embodiments 1 to 57, the conjugate of embodiment 58or 59, or the composition of any one of embodiments 60 to 63 to thesubject.

E77. A method for treating a seizure disorder in a subject in needthereof, comprising administering an effective amount of the oligomer ofany one embodiments 1 to 57, the conjugate of embodiment 58 or 59, orthe composition of any one of embodiments 60 to 61 to the subject.

E78. The method of embodiment 77, wherein the seizure disorder is adisease selected from epilepsy, juvenile myoclonic epilepsy, reflexepilepsy, benign familial infantile epilepsy (BFIE), infantileconvulsions, infantile spasms, choreoathetosis (ICCA) syndrome,injury-associated seizures, brain injury, brain strokes, meningitis, andfebrile seizures.

E79. A method for treating or preventing a neurological disordercomprising administering an effective amount of the oligomer of any oneof embodiments 1 to 57, the conjugate of embodiment 58 or 59, or thecomposition of any one of embodiments 60 to 63.

E80. The method of embodiment 79, wherein the neurological disorder isselected from progressive supranuclear palsy, frontotemporaldementia-tau (FTD-tau), frontotemporal dementia and parkinsonism linkedto chromosome 17 (FTDP-17), corticobasal degeneration (CBD), traumaticbrain injury, chronic traumatic encephalopathy, HIV associatedneurocognitive disorders, Argyrophilic grain disease, Downsyndrome-Alzheimer's disease, Amnestic mild cognitiveimpairment-Alzheimer's disease, Parkinson's disease dementia,Hallervorden-Spatz disease (Pantothenate kinase-associatedneurodegeneration), Niemann Pick disease type C, Myotonic dystrophy,Amyotrophic lateral sclerosis, Parkinson's disease, Huntington'sdisease, Hemimegalencephaly, Tuberous sclerosis complex, Focal corticaldysplasia type 2b, or Ganglion cell tumors. In certain embodiments, thedisease or condition is an epileptic disorder without tauopathy, e.g.,Dravet Syndrome (severe myoclonic epilepsy of infancy), Temporal lobeepilepsy, Ohtahara syndrome (early infantile epileptic encephalopathywith suppression bursts), Lafora body disease, Generalized epilepsy withfebrile seizures, Infantile spasms (West syndrome), Lennox Gastautsyndrome, Angelman Syndrome, Rett Syndrome, Landau Kleffner syndrome,focal seizures, simple focal seizures (no loss of consciousness), focaldyscognitive seizures (impairment of consciousness), focal seizureevolving to generalized tonic-clonic (GTC) convulsions, generalizedseizures (convulsive or non-convulsive with bilateral dischargesinvolving subcortical structures), absence seizures, myoclonic seizures,clonic seizures, tonic seizures, tonic-clonic seizures, atonic seizures,an autistic disorder, an autism spectrum disorder (e.g., as defined inthe Diagnostic and Statistical Manual of Mental Disorders V (DSM-V)), anAsperger's disorder, a pervasive developmental disorder, and anycombination thereof.

E81. The method of any one of embodiments 71 to 80, wherein the subjectis a human.

E82. Use of the oligomer according to any one of the embodiments 1 to 57for the manufacture of a medicament for the treatment of a neurologicaldisorder.

E83. The oligomer of any one of embodiments 1 to 57 for use in therapyof a disease or condition.

E84. The oligomer for use of embodiment 83, wherein the disease orcondition is a neurological disorder.

E85. The oligomer of embodiment 50, wherein the oligomer isATtTCcaaattcactTTtAC (SEQ ID NO: 472) with the chemical structure ofOxyAs OxyTs DNAts OxyTs OxyMCs DNAcs DNAas DNAas DNAas DNAts DNAts DNAcsDNAas DNAcs DNAts OxyTs OxyTs DNAts OxyAs OxyMC (ASO-001940);AtTTCcaaattcactTTtAC (SEQ ID NO: 473) with the chemical structure ofOxyAs DNAts OxyTs OxyTs OxyMCs DNAcs DNAas DNAas DNAas DNAts DNAts DNAcsDNAas DNAcs DNAts OxyTs OxyTs DNAts OxyAs OxyMC (ASO-001933);ATTTccaaattcactTTTAC (SEQ ID NO: 474) with the chemical structure ofOxyAs OxyTs OxyTs OxyTs DNAcs DNAcs DNAas DNAas DNAas DNAts DNAts DNAcsDNAas DNAcs DNAts OxyTs OxyTs OxyTs OxyAs OxyMC (ASO-001919);ATTTCcaaattcacttTTAC (SEQ ID NO: 482) with the chemical structure ofOxyAs OxyTs DNAts OxyTs DNAcs OxyMCs DNAas OxyAs DNAas DNAts DNAts DNAcsDNAas DNAcs DNAts OxyTs DNAts OxyTs OxyAs OxyMC (ASO-001962);ATTtCcaaattcacTtTtAC (SEQ ID NO: 487) with the chemical structure ofOxyAs OxyTs OxyTs DNAts OxyMCs DNAcs DNAas DNAas DNAas DNAts DNAts DNAcsDNAas DNAcs OxyTs DNAts OxyTs DNAts OxyAs OxyMC (ASO-002038);AtTTCcaaattcactTTtAC (SEQ ID NO: 493) with the chemical structure ofOxyTs OxyMCs OxyMCs OxyAs OxyAs DNAas DNAts DNAts DNAcs DNAas DNAcsDNAts DNAts DNAts OxyTs OxyAs OxyMCs (ASO-002439); ATtTCcaaattcactTTtAC(SEQ ID NO: 524) with the chemical structure of OxyAs OxyTs DNAts OxyTsDNAcs OxyMCs DNAas DNAas DNAas DNAts DNAts DNAcs DNAas DNAcs DNAts OxyTsOxyTs DNAts OxyAs OxyMCs (ASO-002263); TATTTccaaattcactTTTA (SEQ ID NO:532) with the chemical structure of OxyTs OxyAs OxyTs OxyTs OxyTs DNAcsDNAcs DNAas DNAas DNAas DNAts DNAts DNAcs DNAas DNAcs DNAts OxyTs OxyTsOxyTs OxyA (ASO-001954); TTaTTtccaaattcaCTtTT (SEQ ID NO: 593) with thechemical structure of OxyTs OxyTs DNAas OxyTs OxyTs DNAts DNAcs DNAcsDNAas DNAas DNAas DNAts DNAts DNAcs DNAas OxyMCs OxyTs DNAts OxyTs OxyT(ASO-001941); TTTATttccaaattcACTTT (SEQ ID NO: 644) with the chemicalstructure of OxyTs OxyTs OxyTs OxyAs OxyTs DNAts DNAts DNAcs DNAcs DNAasDNAas DNAas DNAts DNAts DNAcs OxyAs OxyMCs OxyTs OxyTs OxyT(ASO-000756); TTtATttccaaattcACtTT (SEQ ID NO: 645) with the chemicalstructure of OxyTs OxyTs DNAts OxyAs OxyTs DNAts DNAts DNAcs DNAcs DNAasDNAas DNAas DNAts DNAts DNAcs OxyAs OxyMCs DNAts OxyTs OxyT(ASO-001967); TTTATttccaaattcaCTTT (SEQ ID NO: 646) with the chemicalstructure of OxyTs OxyTs OxyTs OxyAs OxyTs DNAts DNAts DNAcs DNAcs DNAasDNAas DNAas DNAts DNAts DNAcs DNAas OxyMCs OxyTs OxyTs OxyT(ASO-001955); TTTAtttccaaattcACTTT (SEQ ID NO: 647) with the chemicalstructure of OxyTs OxyTs OxyTs OxyAs DNAts DNAts DNAts DNAcs DNAcs DNAasDNAas DNAas DNAts DNAts DNAcs OxyAs OxyMCs OxyTs OxyTs OxyT(ASO-001948); CTTTAtttccaaattCACTT (SEQ ID NO: 677) with the chemicalstructure of OxyMCs OxyTs OxyTs OxyTs OxyAs DNAts DNAts DNAts DNAcsDNAcs DNAas DNAas DNAas DNAts DNAts OxyMCs OxyAs OxyMCs OxyTs OxyT(ASO-000757); CTTTAtttccaaattcACTT (SEQ ID NO: 679) with the chemicalstructure of OxyMCs OxyTs OxyTs OxyTs OxyAs DNAts DNAts DNAts DNAcsDNAcs DNAas DNAas DNAas DNAts DNAts DNAcs OxyAs OxyMCs OxyTs OxyT(ASO-001928); ACTTTatttccaaattCACTT (SEQ ID NO: 680) with the chemicalstructure of OxyAs OxyMCs OxyTs OxyTs OxyTs DNAas DNAts DNAts DNAtsDNAcs DNAcs DNAas DNAas DNAas DNAts DNAts OxyMCs OxyAs OxyMCs OxyTs OxyT(ASO-001968); CTTTatttccaaattCACTT (SEQ ID NO: 681) with the chemicalstructure of OxyMCs OxyTs OxyTs OxyTs DNAas DNAts DNAts DNAts DNAcsDNAcs DNAas DNAas DNAas DNAts DNAts OxyMCs OxyAs OxyMCs OxyTs OxyT(ASO-001921); CTtTAtttccaaattCAcTT (SEQ ID NO: 683) with the chemicalstructure of OxyMCs OxyTs DNAts OxyTs OxyAs DNAts DNAts DNAts DNAcsDNAcs DNAas DNAas DNAas DNAts DNAts OxyMCs OxyAs DNAcs OxyTs OxyT(ASO-001942); TTTccaaattcaCTT (SEQ ID NO: 684) with the chemicalstructure of OxyTs OxyTs OxyTs DNAcs DNAcs DNAas DNAas DNAas DNAts DNAtsDNAcs DNAas OxyMCs OxyTs OxyT (ASO-000128); CtTTAtttccaaattCAcTT (SEQ IDNO: 685) with the chemical structure of OxyMCs DNAts OxyTs OxyTs OxyAsDNAts DNAts DNAts DNAcs DNAcs DNAas DNAas DNAas DNAts DNAts OxyMCs OxyAsDNAcs OxyTs OxyT (ASO-001935); ATTtccaaattcaCTT (SEQ ID NO: 686) withthe chemical structure of OxyAs OxyTs OxyTs DNAts DNAcs DNAcs DNAasDNAas DNAas DNAts DNAts DNAcs DNAas OxyMCs OxyTs OxyT (ASO-000013);CTTtatttccaaatTcACT (SEQ ID NO: 714) with the chemical structure ofOxyMCs OxyTs OxyTs DNAts DNAas DNAts DNAts DNAts DNAcs DNAcs DNAas DNAasDNAas DNAts OxyTs DNAcs OxyAs OxyMCs OxyT (ASO-002012);ACTTTatttccaaattCACT (SEQ ID NO: 715) with the chemical structure ofOxyAs OxyMCs OxyTs OxyTs OxyTs DNAas DNAts DNAts DNAts DNAcs DNAcs DNAasDNAas DNAas DNAts DNAts OxyMCs OxyAs OxyMCs OxyT (ASO-001962);ACTTtatttccaaatTCACT (SEQ ID NO: 716) with the chemical structure ofOxyAs OxyMCs OxyTs OxyTs DNAts DNAas DNAts DNAts DNAts DNAcs DNAcs DNAasDNAas DNAas DNAts OxyTs OxyMCs OxyAs OxyMCs OxyT (ASO-001956),CTTtatttccaaatTCACT (SEQ ID NO: 720) with the chemical structure ofOxyMCs OxyTs OxyTs DNAts DNAas DNAts DNAts DNAts DNAcs DNAcs DNAas DNAasDNAas DNAts OxyTs OxyMCs OxyAs OxyMCs OxyT (ASO-1995);CTTtatttccaaatTCAC (SEQ ID NO: 740) with the chemical structure ofOxyMCs OxyTs OxyTs DNAts DNAas DNAts DNAts DNAts DNAcs DNAcs DNAas DNAasDNAas DNAts OxyTs OxyMCs OxyAs OxyMC (ASO-002007); CTTtatttccaaatTcAC(SEQ ID NO: 745) with the chemical structure of OxyMCs OxyTs OxyTs DNAtsDNAas DNAts DNAts DNAts DNAcs DNAcs DNAas DNAas DNAas DNAts OxyTs DNAcsOxyAs OxyMC (ASO-001997); AATaactttatttCCA (SEQ ID NO: 773) with thechemical structure of OxyAs OxyAs OxyTs DNAas DNAas DNAcs DNAts DNAtsDNAts DNAas DNAts DNAts DNAts OxyMCs OxyMCs OxyA (ASO-000118);AGTaataactttaTTT(SEQ ID NO: 780) with the chemical structure of OxyAsOxyGs OxyTs DNAas DNAas DNAts DNAas DNAas DNAcs DNAts DNAts DNAts DNAasOxyTs OxyTs OxyT (ASO-000170); AGTaataactttATT (SEQ ID NO: 782) with thechemical structure of OxyAs OxyGs OxyTs DNAas DNAas DNAts DNAas DNAasDNAcs DNAts DNAts DNAts OxyAs OxyTs OxyT (ASO-000125); AGAgtaataacttTAT(SEQ ID NO: 784) with the chemical structure of OxyAs OxyGs OxyAs DNAgsDNAts DNAas DNAas DNAts DNAas DNAas DNAcs DNAts DNAts OxyTs OxyAs OxyT(ASO-000134); AGAgtaataactTTA (SEQ ID NO: 786) with the chemicalstructure of OxyAs OxyGs OxyAs DNAgs DNAts DNAas DNAas DNAts DNAas DNAasDNAcs DNAts OxyTs OxyTs OxyA (ASO-000178); AATcagagtaatAAC (SEQ ID NO:794) with the chemical structure of OxyAs OxyAs OxyTs DNAcs DNAas DNAgsDNAas DNAgs DNAts DNAas DNAas DNAts OxyAs OxyAs OxyMC (ASO-000307);TTAatcagagtaaTAA (SEQ ID NO: 795) with the chemical structure of OxyTsOxyTs OxyAs DNAas DNAts DNAcs DNAas DNAgs DNAas DNAgs DNAts DNAas DNAasOxyTs OxyAs OxyA (ASO-000204); TAAtcagagtaaTAA (SEQ ID NO: 796) with thechemical structure of OxyTs OxyAs OxyAs DNAts DNAcs DNAas DNAgs DNAasDNAgs DNAts DNAas DNAas OxyTs OxyAs OxyA (ASO-000330); TTTaatcagagtaATA(SEQ ID NO: 797) with the chemical structure of OxyTs OxyTs OxyTs DNAasDNAas DNAts DNAcs DNAas DNAgs DNAas DNAgs DNAts DNAas OxyAs OxyTs OxyA(ASO-000326); or TTTaatcagagtAAT (SEQ ID NO: 798) with the chemicalstructure of OxyTs OxyTs OxyTs DNAas DNAas DNAts DNAcs DNAas DNAgs DNAasDNAgs DNAts OxyAs OxyAs OxyT (ASO-000249).

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A to 1C show Tau genomic, mRNA, and protein sequences. SEQ ID NO:1 in FIG. 1A represents a MAPT genomic sequence. SEQ ID NO: 1 isidentical to a MAPT pre-mRNA sequence except that nucleotide “t” in SEQID NO: 1 is shown as “u” in pre-mRNA. SEQ ID NO: 2 in FIG. 1B representsa MAPT mRNA sequence except that nucleotide “t” in SEQ ID NO: 2 is shownas “u” in mRNA. The Tau protein sequence encoded by the MAPT mRNA isshown as SEQ ID NO: 3 in FIG. 1C. FIG. 2 shows exemplary oligomers,designs (ASO Sequence), and chemical structure of the oligomers.

FIG. 2 lists the oligomer name, antisense oligomer (ASO) identificationnumber, ASO sequence, SEQ ID Number, target start and end positions onthe MAPT pre-mRNA sequence and chemical structure. Examples of oligomerswith mismatched bases are provided in FIG. 2 as “mm.” The specificmismatched base-pairs are bolded, underlined, italicized, andhighlighted.

FIG. 3 shows exemplary oligomers targeting nucleotides 134,947 to138,940 of SEQ ID NO: 1. FIG. 3 lists the SEQ ID number, oligomer name,ASO identification number, ASO sequence, target start and end positionson the MAPT pre-mRNA sequence, target start on the mature mRNA sequenceand normalized Tau/Tuj-1 and Tuj-1 immunocytochemistry values (asdiscussed in Example 2 below). Examples of oligomers with mismatchedbases are provided in FIG. 3 as “mm.” The specific mismatched base-pairsare bolded, underlined, italicized, and highlighted.

FIG. 4 is a graph demonstrating primary neuronal spontaneous calciumoscillations. Primary neuronal spontaneous calcium oscillations weremeasured as described previously (Murphy et.al., 1992, J. Neurosci.12:4834-4845). Addition of theα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorantagonist, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 3 μM), reducedcalcium oscillations by 20% representing the total AMPA response in theassay (AMPA labeled bar shown). Calcium oscillations were reducedfurther, by about 80%, when N-methyl-D-aspartate (NMDA) receptorfunction was blocked by 1 mM MgCl₂ (NMDA labeled bar shown).

FIG. 5 is a graph showing inhibition of AMPA mediated calciumoscillations by antisense oligomers as an indication of neuronal networkactivity disruption. Antisense oligomer inhibition of spontaneouscalcium oscillations mediated by either NMDA or AMPA was assessed in thepresence or absence of 1 mM MgCl₂ (representing 100% control in eachcase). Addition of 25 μM antisense oligomers (TGTgatgcaggaGTT) (SEQ IDNO: 304) (ASO-00007) inhibited AMPA receptor but not NMDA receptormediated oscillations. The ASO and other oligomers that behavedsimilarly were shown to negatively impact central nervous system (CNS)network activity in vivo and electrophysiologic spontaneous neuronalactivity in vitro (data not shown).

FIG. 6 shows the impact of Tau antisense oligonucleotides on spontaneouscalcium oscillations in primary neurons. FIG. 6 lists the ASOidentification number, ASO sequence, SEQ ID Number, target start and endpositions on the MAPT pre-mRNA sequence, calcium oscillation data as apercent of control (as discussed in Example 3 below) and IC₅₀ values ofTau neurons (as discussed in Example 2 below). Examples of oligomerswith mismatched bases are provided in FIG. 6 as “mm.” The specificmismatched base-pairs are bolded, underlined, italicized, andhighlighted. FIG. 7 shows the in vivo tolerability of exemplaryoligomers.

FIG. 7 lists the ASO identification number, ASO sequence, SEQ ID Number,target start and end positions on the MAPT pre-mRNA sequence, in vivoacute tolerability score (as discussed in Example 5 below) and thepercent of brain MAPT mRNA remaining after administration (as alsodiscussed in Example 5 below).

FIG. 8A shows correlation analysis of sequence score vs. in vivotolerability score. Sequence score for each oligomer was calculated byinserting appropriate numbers in the formula: ((number of C nucleotidesor its analogs—number of G nucleotides)/nucleotide length (i.e.,number)). In vivo tolerability scores were calculated based uponobservations following a single intra-cerebroventricular (ICV)administration of 100 μg oligomers in mice or intrathecal (i.t.)administration of 900 μg or up to 1500 μg oligomers in rats. The rodentswere observed under five categories: 1) hyperactivity; 2) decreasedactivity and arousal; 3) motor dysfunction and/or ataxia; 4) abnormalposture and breathing; and 5) tremor and/or convulsions. The total invivo tolerability score is the sum of five unit scores; each of the unitscores is measured on a scale of 0-4. Therefore, the total score of invivo tolerability can range from 0 to 20. The sequence score calculatedby the formula is on the X-axis, and the in vivo tolerability score ison the Y-axis.

FIG. 8B shows correlation of in vitro potency (Y-axis) and in vivo TaumRNA reduction (X-axis). In vitro potency (IC₅₀) was correlated with invivo Tau mRNA reduction following administration of 100 μg ASOs, 2 weekspost-dose in mice (r²=0.54; p<0.001). Squares represent oligomersprioritized based on the in vitro Tau protein reduction and primaryneuronal health as assessed by tubulin and spontaneous calciumoscillations (FIGS. 3, 4, 6, and 7).

FIGS. 9A-9B are graphs showing brain Tau mRNA (9A) and Tau protein (9B)reduction over time following a single ICV bolus of 100 μg ASO-000013(i.e., ATTtccaaattcaCTT, i.e., SEQ ID NO: 686 in which the upper caseletters represent LNA nucleotides while the lower case letters representDNA nucleotides) administration into wild type C57 mice (N=12). Tau mRNAexpression (normalized to GAPDH) was measured at 2, 4, 8 and 12 weekspost injection. Tau protein (% of saline) level was measured at 2, 4, 8and 12 weeks post injection. (* p<0.01, ***p,0.001) Both Tau mRNA andprotein returned to baseline at 20 weeks post-dose (data not shown).

FIG. 10 is a graph showing that brain concentrations of ASO-000013 weredetected up to 12 weeks following administration of a single ICV bolusof 100 μg into wild type C57 mice (N=12).

FIG. 11 is a graph showing brain Tau mRNA reduction following 3 day or 4week post 300 μg single bolus intrathecal (IT) administration ofoligomers (ASO-000013 and ASO-000757 (i.e., CTTTAtttccaaattCACTT (SEQ IDNO: 677)) in rat (N=6).

FIG. 12 shows the comparison of the sequence of selected oligomers andthe sequence of Rho A which aligns with a portion of the MAPT genomicsequence (SEQ ID NO: 1). The RhoA sequence is listed asactttatttccaaatacacttcttt (SEQ ID NO: 959). The mismatches between theselected oligomers and the Rho A sequence were highlighted. The sequenceof ASO-000757 has one mismatch compared to the corresponding RhoAsequence; the sequences of ASO-0001967, ASO-000755, and ASO-001941 havetwo mismatches compared to the corresponding RhoA sequence; and thesequences of ASO-000753, ASO-002038, ASO-001933, and ASO-001940 havefour mismatches compared to the corresponding RhoA sequence. FIG. 12shows that the traditional gapmers (i.e., ASO-000757, ASO-000755, andASO-000753) are not tolerated beyond 4 weeks following a single 100 μgICV bolus dose while the alternating flank gapmers (i.e., ASO-001941,ASO-002038, ASO-001933, and ASO-001940) exhibit tolerability beyond 4weeks. Tubulin inhibition was highly correlated, in this data set, tolong term tolerability. Rho A reduction greater than 25% (i.e.,ASO-000757, ASO-000755, and ASO-000753) was also correlated with lack oflong term tolerability (greater than 4 weeks following a single ICVbolus injection of 100 μg of each ASO shown).

FIG. 13 shows that ASO-001933 produces dose responsive brain hTauprotein reduction after a single ICV injection in hTau mouse brain.Saline or 50, 100, 150 or 200 μg of Tau ASO was injected ICV in hTaumice (n=10 per group). X-axis shows the dose of ASO-001933, and theY-axis shows the hTau protein expression after the ASO injectioncompared to the hTau protein expression after the saline injection (% ofsaline).

FIGS. 14A and 14B show that Tau ASO-000774 mediated insoluble andsoluble Tau reduction rescued hyperactivity in a mouse model oftauopathy (Tg4510). Tau reduction reverses hyperactivity in Tg4510 micein running wheel assay. FIG. 14A shows that a single 100 μg ICV bolusreduces total Tau protein using BT-2 and HT-7 ELISA. The left panelshows the total Tau protein expression (% of control) when a vehicle isadministered (i.e., 100%), and the right panel shows the total Tauprotein expression when ASO-000774 was administered. FIG. 14B shows thetotal wheel counts assessed in a running wheel assay in Tg4510(tauopathy mouse model) and double negative littermate controls (DblNeg) as described in the Example 7.

FIGS. 15A and 15B show that tau oligomers (e.g., ASO-000762) can rescuepremature lethality and reduced tonic clonic seizure in a mouse model ofDravet Syndrome, respectively. FIG. 15A shows survival plots of Dravetmice and littermate controls treated with a single ICV administration of20 or 37 μg of Tau ASO-000762 targeting the 3′-UTR region of Tau mRNA.The oligomer has been shown to reduce 20-50% of Tau protein (data notshown) at 10 days postnatally. The upper line in FIG. 15A shows thepercent survival of the Dravet mice treated with a single ICVadministration of 37 μg of Tau ASO-000762. The middle line in FIG. 15Ashows the percent survival of the Dravet mice treated with a single ICVadministration of 20 μg of Tau ASO-000762. The lower line in FIG. 15Ashows the percent survival of the Dravet mice treated with a single ICVadministration of saline. FIG. 15B shows the percent mice withouthyperthermia-induced Generalized Tonic-Clonic Seizures (GTCS) in Dravetmice. The GTCS was measured 8-9 weeks post-injection of ASO-000762 at 20μg. The percent mice without GTCS after administration of vehicle isshown in circle, and the percent mice without GTCS after administrationof ASO-000762 is shown as square.

FIGS. 16A and 16B show exemplary oligomers, designs, and their chemicalstructures. FIG. 16A lists the antisense oligomer (ASO) identificationnumber, SEQ ID number, ASO sequence, target start and end positions onthe Tau pre-mRNA sequence, IC₅₀ values of Tau neurons (as discussed inExample 8 below) and percent Tau inhibition (as also discussed inExample 8 below). FIG. 16B shows the specific chemical structure of theoligomers shown in FIG. 16A and lists the antisense oligomer (ASO)identification number, ASO sequence, target start and end positions onthe Tau pre-mRNA sequence and chemical structure.

FIG. 17A is an image of brain regions showing pathologic Tauaccumulation in PSP.

FIG. 17B shows regional Tau mRNA changes in a control monkey (left) orin a monkey that had received two single 16 mg intrathecal bolus dosesof ASO-001933, one week apart (right). The Tau mRNA changes wereassessed two weeks post-dosing by fluorescence in situ hybridization(FISH) using Tau mRNA specific probes in substantia nigra, pontinenucleus and central cerebellar dentate nucleus. Tau mRNA accumulation isshown as lighter shades.

FIG. 18A shows Tau protein reduction in brain following intrathecaldosing of ASO-001933 in nonhuman primates (NHPs). Regional Tau mRNAchanges in a control monkey or a monkey that had received two single 8mg intrathecal bolus doses of ASO-001933, two weeks apart, were assessed4, 8, or 12 weeks post-dosing by Tau ELISAs (BT2/HT7 or Tau12/BT2). Theregional Tau mRNA changes were measured in pons, cerebellum (CBL),parietal cortex (ParC), frontal cortex (FrC), occipital cortex (OccC),temporal cortex (TemC), and hippocampus (Hipp).

FIG. 18B shows Tau protein reduction in cerebrospinal fluid (CSF)following intrathecal dosing of ASO-001933 in nonhuman primates (NHPs).Y-axis shows percent baseline of Tau protein reduction in CSF, andX-axis shows weeks post last dose.

FIG. 19A shows that ASO-002038 (Tau ASO) produces durable, doseresponsive brain hTau mRNA reduction after a singleintracerebroventricular (ICV) injection in hTau mouse brain. Saline or25, 50,100, and 150 μg of Tau ASO was injected ICV in hTau mice (n=10per group). The frontal cortical region was dissected 1 week post doseto determine total Tau mRNA levels by qRT-PCR. 1-way ANOVA analysis wasused ***p<0.001. Error bars represent mean+/−SEM.

FIG. 19B shows that ASO-002038 (Tau ASO) produces durable, doseresponsive brain hTau mRNA reduction after a single intrathecal (IT)injection in surgical lumbar catheterized rats. Saline or 400, 900, and1500 μg of Tau ASO was injected IT in rats (n=10 per group). The frontalcortical region was dissected 1 week post dose to determine total TaumRNA levels by qRT-PCR. 1-way ANOVA analysis was used ***p<0.001. Errorbars represent mean+/−SEM.

FIGS. 20A and 20B show exemplary oligomers, designs, and chemicalstructures tested by Quantigene® analysis. FIG. 20A lists the antisenseoligomer (ASO) identification number, SEQ ID number, ASO sequence,target start and end positions on the Tau pre-mRNA sequence, startposition on the mature mRNA sequence, and Quantigene® expression of mRNA(as discussed in Example 10 below). FIG. 20B shows the specific chemicalstructure of the oligomers shown in FIG. 20A and lists the antisenseoligomer (ASO) identification number, ASO sequence, target start and endpositions on the Tau pre-mRNA sequence and chemical structure.

DETAILED DESCRIPTION OF INVENTION

I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleotide sequence,” is understood torepresent one or more nucleotide sequences. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleotidesequences are written left to right in 5′ to 3′ orientation. Amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects ofthe disclosure, which can be had by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

The term “about” is used herein to mean approximately, roughly, around,or in the regions of. When the term “about” is used in conjunction witha numerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” can modify a numerical value above and below the stated value bya variance of, e.g., 10 percent, up or down (higher or lower). Forexample, if it is stated that “the oligomer reduces expression of Tauprotein in a cell following administration of the oligomer by at leastabout 60%,” it is implied that the Tau levels are reduced by a range of50% to 70%.

The term “nucleic acids” or “nucleotides” is intended to encompassplural nucleic acids. In some embodiments, the term “nucleic acids” or“nucleotides” refers to a target sequence, e.g., pre-mRNAs, mRNAs, orDNAs in vivo or in vitro. When the term refers to the nucleic acids ornucleotides in a target sequence, the nucleic acids or nucleotides canbe naturally occurring sequences within a cell. In other embodiments,“nucleic acids” or “nucleotides” refers to a sequence in the oligomersof the invention. When the term refers to a sequence in the oligomers,the nucleic acids or nucleotides are not naturally occurring. In oneembodiment, the nucleic acids or nucleotides in the oligomers areproduced synthetically or recombinantly, but are not a naturallyoccurring sequence or a fragment thereof. In another embodiment, thenucleic acids or nucleotides in the oligomers contain at least onenucleotide analog that is not naturally occurring in nature. The term“nucleic acid” or “nucleoside” refers to a single nucleic acid segment,e.g., a DNA, an RNA, or an analog thereof, present in a polynucleotide.“Nucleic acid” or “nucleoside” includes naturally occurring nucleicacids or non-naturally occurring nucleic acids. In some embodiments, theterms “nucleotide”, “unit” and “monomer” are used interchangeably. Itwill be recognized that when referring to a sequence of nucleotides ormonomers, what is referred to is the sequence of bases, such as A, T, G,C or U, and analogs thereof.

The term “nucleotide” as used herein, refers to a glycoside comprising asugar moiety, a base moiety and a covalently linked group (linkagegroup), such as a phosphate or phosphorothioate internucleotide linkagegroup, and covers both naturally occurring nucleotides, such as DNA orRNA, and non-naturally occurring nucleotides comprising modified sugarand/or base moieties, which are also referred to as “nucleotide analogs”herein. Herein, a single nucleotide (unit) can also be referred to as amonomer or nucleic acid unit. In certain embodiments, the term“nucleotide analogs” refers to nucleotides having modified sugarmoieties. Non-limiting examples of the nucleotides having modified sugarmoieties (e.g., LNA) are disclosed elsewhere herein. In otherembodiments, the term “nucleotide analogs” refers to nucleotides havingmodified base moieties. The nucleotides having modified base moietiesinclude, but are not limited to, 5-methylcytosine, isocytosine,pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

The term “nucleoside” as used herein is used to refer to a glycosidecomprising a sugar moiety and a base moiety, and can therefore be usedwhen referring to the nucleotide units, which are covalently linked bythe internucleotide linkages between the nucleotides of the oligomer. Inthe field of biotechnology, the term “nucleotide” is often used to referto a nucleic acid monomer or unit, and as such in the context of anoligonucleotide can refer to the base—such as the “nucleotide sequence”,typically refer to the nucleobase sequence (i.e. the presence of thesugar backbone and internucleoside linkages are implicit). Likewise,particularly in the case of oligonucleotides where one or more of theinternucleoside linkage groups are modified, the term “nucleotide” canrefer to a “nucleoside” for example the term “nucleotide” can be used,even when specifying the presence or nature of the linkages between thenucleosides.

The term “nucleotide length” as used herein means the total number ofthe nucleotides (monomers) in a given sequence. For example, thesequence of AAAgatgaaatttgctcTTA (SEQ ID NO: 4) has 20 nucleotides; thusthe nucleotide length of the sequence is 20. The term “nucleotidelength” is therefore used herein interchangeably with “nucleotidenumber.”

As one of ordinary skill in the art would recognize, the 5′ terminalnucleotide of an oligonucleotide does not comprise a 5′ internucleotidelinkage group, although it can comprise a 5′ terminal group.

As used herein, a “coding region” or “coding sequence” is a portion ofpolynucleotide which consists of codons translatable into amino acids.Although a “stop codon” (TAG, TGA, or TAA) is typically not translatedinto an amino acid, it can be considered to be part of a coding region,but any flanking sequences, for example promoters, ribosome bindingsites, transcriptional terminators, introns, untranslated regions(“UTRs”), and the like, are not part of a coding region. The boundariesof a coding region are typically determined by a start codon at the 5′terminus, encoding the amino terminus of the resultant polypeptide, anda translation stop codon at the 3′ terminus, encoding the carboxylterminus of the resulting polypeptide.

The term “non-coding region” as used herein means a nucleotide sequencethat is not a coding region. Examples of non-coding regions include, butare not limited to, promoters, ribosome binding sites, transcriptionalterminators, introns, untranslated regions (“UTRs”), non-coding exonsand the like. Some of the exons can be wholly or part of the 5′untranslated region (5′ UTR) or the 3′ untranslated region (3′ UTR) ofeach transcript. The untranslated regions are important for efficienttranslation of the transcript and for controlling the rate oftranslation and half-life of the transcript.

The term “region” when used in the context of a nucleotide sequencerefers to a section of that sequence. For example, the phrase “regionwithin a nucleotide sequence” or “region within the complement of anucleotide sequence” refers to a sequence shorter than the nucleotidesequence, but longer than at least 10 nucleotides located within theparticular nucleotide sequence or the complement of the nucleotidessequence, respectively. The term “sub-sequence” or “subsequence” canalso refer to a region of a nucleotide sequence.

The term “downstream,” when referring to a nucleotide sequence, meansthat a nucleic acid or a nucleotide sequence is located 3′ to areference nucleotide sequence. In certain embodiments, downstreamnucleotide sequences relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to a reference nucleotide sequence.

As used herein, the term “regulatory region” refers to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding region, and whichinfluence the transcription, RNA processing, stability, or translationof the associated coding region. Regulatory regions can includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites,UTRs, and stem-loop structures. If a coding region is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding sequence.

The term “transcript” as used herein can refer to a primary transcriptthat is synthesized by transcription of DNA and becomes a messenger RNA(mRNA) after processing, i.e., a precursor messenger RNA (pre-mRNA), andthe processed mRNA itself. The term “transcript” can be interchangeablyused with “pre-mRNA” and “mRNA.” After DNA strands are transcribed toprimary transcripts, the newly synthesized primary transcripts aremodified in several ways to be converted to their mature, functionalforms to produce different proteins and RNAs such as mRNA, tRNA, rRNA,lncRNA, miRNA and others. Thus, the term “transcript” can include exons,introns, 5′ UTRs, and 3′ UTRs.

The term “expression” as used herein refers to a process by which apolynucleotide produces a gene product, for example, a RNA or apolypeptide. It includes, without limitation, transcription of thepolynucleotide into messenger RNA (mRNA) and the translation of an mRNAinto a polypeptide. Expression produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation or splicing, or polypeptides with post translationalmodifications, e.g., methylation, glycosylation, the addition of lipids,association with other protein subunits, or proteolytic cleavage.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids refer to two or more sequences that are the same orhave a specified percentage of nucleotides or amino acid residues thatare the same, when compared and aligned (introducing gaps, if necessary)for maximum correspondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid or nucleotidesequences.

One such non-limiting example of a sequence alignment algorithm is thealgorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci.,87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad.Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs(Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certainembodiments, Gapped BLAST can be used as described in Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul etal., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2(Genentech, South San Francisco, Calif.) or Megalign (DNASTAR) areadditional publicly available software programs that can be used toalign sequences. In certain embodiments, the percent identity betweentwo nucleotide sequences is determined using the GAP program in the GCGsoftware package (e.g., using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). Incertain alternative embodiments, the GAP program in the GCG softwarepackage, which incorporates the algorithm of Needleman and Wunsch (J.Mol. Biol. (48): 444-453 (1970)) can be used to determine the percentidentity between two amino acid sequences (e.g., using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certainembodiments, the percent identity between nucleotide or amino acidsequences is determined using the algorithm of Myers and Miller (CABIOS,4:11-17 (1989)). For example, the percent identity can be determinedusing the ALIGN program (version 2.0) and using a PAM120 with residuetable, a gap length penalty of 12 and a gap penalty of 4. One skilled inthe art can determine appropriate parameters for maximal alignment byparticular alignment software. In certain embodiments, the defaultparameters of the alignment software are used.

In certain embodiments, the percentage identity “X” of a firstnucleotide sequence to a second nucleotide sequence is calculated as100×(Y/Z), where Y is the number of amino acid residues scored asidentical matches in the alignment of the first and second sequences (asaligned by visual inspection or a particular sequence alignment program)and Z is the total number of residues in the second sequence. If thelength of a first sequence is longer than the second sequence, thepercent identity of the first sequence to the second sequence will behigher than the percent identity of the second sequence to the firstsequence.

Different regions within a single polynucleotide target sequence thatalign with a polynucleotide reference sequence can each have their ownpercent sequence identity. It is noted that the percent sequenceidentity value is rounded to the nearest tenth. For example, 80.11,80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16,80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted thatthe length value will always be an integer.

As used herein, the terms “homologous” and “homology” areinterchangeable with the terms “identity” and “identical.”

The term “naturally occurring variant thereof” refers to variants of theTau polypeptide sequence or MAPT nucleic acid sequence (e.g.,transcript) which exist naturally within the defined taxonomic group,such as mammalian, such as mouse, monkey, and human. Typically, whenreferring to “naturally occurring variants” of a polynucleotide the termalso can encompass any allelic variant of the MAPT-encoding genomic DNAwhich is found at Chromosomal position 17q21 by chromosomaltranslocation or duplication, and the RNA, such as mRNA derivedtherefrom. “Naturally occurring variants” can also include variantsderived from alternative splicing of the MAPT mRNA. When referenced to aspecific polypeptide sequence, e.g., the term also includes naturallyoccurring forms of the protein, which can therefore be processed, e.g.,by co- or post-translational modifications, such as signal peptidecleavage, proteolytic cleavage, glycosylation, etc.

In determining the degree of “complementarity” between oligomers of theinvention (or regions thereof) and the target region of the nucleic acidwhich encodes mammalian Tau (e.g., the MAPT gene), such as thosedisclosed herein, the degree of “complementarity” (also, “homology” or“identity”) is expressed as the percentage identity (or percentagehomology) between the sequence of the oligomer (or region thereof) andthe sequence of the target region (or the reverse complement of thetarget region) that best aligns therewith. The percentage is calculatedby counting the number of aligned bases that are identical between thetwo sequences, dividing by the total number of contiguous monomers inthe oligomer, and multiplying by 100. In such a comparison, if gapsexist, it is preferable that such gaps are merely mismatches rather thanareas where the number of monomers within the gap differs between theoligomer of the invention and the target region.

The term “complement” as used herein indicates a sequence that iscomplementary to a reference sequence. It is well known thatcomplementarity is the base principle of DNA replication andtranscription as it is a property shared between two DNA or RNAsequences, such that when they are aligned antiparallel to each other,the nucleotide bases at each position in the sequences will becomplementary, much like looking in the mirror and seeing the reverse ofthings. Therefore, for example, the complement of a sequence of 5′“ATGC”3′ can be written as 3′ “TACG”5′ or 5′ “GCAT”3′. The terms“reverse complement”, “reverse complementary” and “reversecomplementarity” as used herein are interchangeable with the terms“complement”, “complementary” and “complementarity.”

The terms “corresponding to” and “corresponds to,” when referencing twoseparate nucleic acid or nucleotide sequences can be used to clarifyregions of the sequences that correspond or are similar to each otherbased on homology and/or functionality, although the nucleotides of thespecific sequences can be numbered differently. For example, differentisoforms of a gene transcript can have similar or conserved portions ofnucleotide sequences whose numbering can differ in the respectiveisoforms based on alternative splicing and/or other modifications. Inaddition, it is recognized that different numbering systems can beemployed when characterizing a nucleic acid or nucleotide sequence(e.g., a gene transcript and whether to begin numbering the sequencefrom the translation start codon or to include the 5′UTR). Further, itis recognized that the nucleic acid or nucleotide sequence of differentvariants of a gene or gene transcript can vary. As used herein, however,the regions of the variants that share nucleic acid or nucleotidesequence homology and/or functionality are deemed to “correspond” to oneanother. For example, a nucleotide sequence of a MAPT transcriptcorresponding to nucleotides X to Y of SEQ ID NO: 1 (“referencesequence”) refers to an MAPT transcript sequence (e.g., MAPT pre-mRNA ormRNA) that has an identical sequence or a similar sequence tonucleotides X to Y of SEQ ID NO: 1. A person of ordinary skill in theart can identify the corresponding X and Y residues in the MAPTtranscript sequence by aligning the MAPT transcript sequence with SEQ IDNO: 1.

The terms “corresponding nucleotide analog” and “correspondingnucleotide” are intended to indicate that the nucleobase in thenucleotide analog and the naturally occurring nucleotide have the samepairing, or hybridizing, ability. For example, when the 2-deoxyriboseunit of the nucleotide is linked to an adenine, the “correspondingnucleotide analog” contains a pentose unit (different from2-deoxyribose) linked to an adenine.

The term “design” or “oligomer design” or “ASO Sequence” as used hereinrefers to a pattern of nucleotides (e.g., DNA) and nucleotide analogs(e.g., LNA) in a given sequence. As used herein, the design of anoligomer is shown by a combination of upper case letters and lower caseletters. For example, an oligomer sequence of tatttccaaattcactttta (SEQID NO: 573) can have oligomer designs of ASO-002350(TAtTTccaaattcactTTTA), ASO-002374 (TAtTTccaaattcacTtTTA), ASO-002386(TATTtccaaattcaCTttTA), ASO-002227 (TATtTccaaattcactTTTA), ASO-002245(TAttTCcaaattcactTTTA), ASO-002261 (TATtTccaaattcacTTtTA), ASO-002276(ATttCcaaattcactTTTA), ASO-002228 (TATTtccaaattcaCtTtTA), ASO-002255(TATTtccaaattcactTTTA), ASO-002285 (TATTtccaaattcacTTtTA), ASO-002230(TATTtccaaattcacTtTTA), ASO-002256 (TATTtccaaattcAcTttTA), or ASO-002279(TATTtccaaattcActTtTA), wherein the upper case letter indicates anucleotide analog (e.g., LNA) and the lower case letter indicates anucleotide (e.g., DNA)

The term “chemical structure” of an oligomer as used herein refers to adetailed description of the components of the oligomers, e.g.,nucleotides (e.g., DNA), nucleotide analogs (e.g., beta-D-oxy-LNA),nucleotide base (e.g., A, T, G, C, U, or MC), and backbone structure(e.g., phosphorothioate or phosphorodiester). For example, a chemicalstructure of ASO-002350 can be OxyTs OxyAs DNAts OxyTs OxyTs DNAcs DNAcsDNAas DNAas DNAas DNAts DNAts DNAcs DNAas DNAcs DNAts OxyTs OxyTs OxyTsOxyAs. FIGS. 2, 16B, and 20B lists non-limiting examples of chemicalstructures that can be applied to any one of the oligomers disclosedherein.

“Potency” is normally expressed as an IC₅₀ or EC₅₀ value, in μM, nM orpM unless otherwise stated. Potency can also be expressed in terms ofpercent inhibition. IC₅₀ is the median inhibitory concentration of atherapeutic molecule. EC₅₀ is the median effective concentration of atherapeutic molecule relative to a vehicle or saline control. Infunctional assays, IC₅₀ is the concentration that reduces a biologicalresponse, e.g., transcription of mRNA or protein expression, by 50% ofthe biological response that is achieved by the therapeutic molecule. Infunctional assays, EC₅₀ is the concentration of a therapeutic moleculethat produces 50% of the biological response, eg., transcription of mRNAor protein expression. IC₅₀ or EC₅₀ can be calculated by any number ofmeans known in the art.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, sports animals, and zoo animalsincluding, e.g., humans, non-human primates, dogs, cats, guinea pigs,rabbits, rats, mice, horses, cattle, bears, and so on.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the composition wouldbe administered. Such composition can be sterile.

An “effective amount” of an oligomer as disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both (1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and (2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. In certainembodiments, a subject is successfully “treated” for a disease orcondition disclosed elsewhere herein according to the methods providedherein if the patient shows, e.g., total, partial, or transientalleviation or elimination of symptoms associated with the disease ordisorder.

II. The Oligomer

The present invention employs oligomeric compounds (referred herein asoligomers), for use in modulating the function of nucleic acid moleculesencoding mammalian Tau, such as the MAPT nucleic acid, e.g., MAPTtranscript, including MAPT pre-mRNA, and MAPT mRNA, or naturallyoccurring variants of such nucleic acid molecules encoding mammalianTau. The term “oligomer” in the context of the present invention, refersto a molecule formed by covalent linkage of two or more nucleotides(i.e., an oligonucleotide).

The oligomer comprises a contiguous nucleotide sequence of from about 10to about 50, such as 10-20, 16-20, 15-25, 10-30, 10-35, 10-40, or 10-45nucleotides in length. The terms “antisense oligomer,” “antisenseoligonucleotide,” and “ASO” as used herein are interchangeable with theterm “oligomer.”

A reference to a SEQ ID number includes a particular nucleobasesequence, but does not include an oligomer design as shown in FIG. 2, 3,6, 7, 16A, 16B, 20A, or 20B. Furthermore, the oligomers disclosed in thefigures herein show a representative design, but are not limited to thespecific design shown in the tables. Herein, a single nucleotide (unit)can also be referred to as a monomer or unit. When this specificationrefers to a specific ASO number (or oligomer name), the referenceincludes the specific oligomer design. For example, when a claim (orthis specification) recites SEQ ID NO: 803, it includes the nucleotidesequence of actttatttccaaattcacttttac. When a claim (or thespecification) recites ASO-002019, it includes the nucleotide sequenceof actttatttccaaattcacttttac with the oligomer design shown in thefigures (i.e., ActtTatttccaaattcactTTtaC). Alternatively, ASO-002019 canbe written as ActtTatttccaaattcactTTtaC, wherein the upper case letteris a modified nucleotide (e.g., LNA) and the lower case letter is anon-modified nucleotide (e.g., DNA). ASO-002019 can also be written asSEQ ID NO: 803, wherein each of the first nucleotide, the fifthnucleotide, the 21^(st) nucleotide, the 22^(nd) nucleotide, and the25^(th) nucleotide from the 5′ end is a modified nucleotide, e.g., LNA,and each of the other nucleotides is a non-modified nucleotide (e.g.,DNA). The oligomers of the invention can also be written as SEQ ID NO:803 with the chemical structure shown in FIG. 2, i.e., OxyAs OxyMCsDNAts DNAts OxyTs DNAas DNAts DNAts DNAts DNAcs DNAcs DNAas DNAas DNAasDNAts DNAts DNAcs DNAas DNAcs DNAts OxyTs OxyTs DNAts DNAas OxyMC.

In various embodiments, the oligomer of the invention does not compriseRNA (units). In some embodiments, the oligomer comprises one or more DNAunits. In one embodiment, the oligomer according to the invention is alinear molecule or is synthesized as a linear molecule. In someembodiments, the oligomer is a single stranded molecule, and does notcomprise short regions of, for example, at least 3, 4 or 5 contiguousnucleotides, which are complementary to equivalent regions within thesame oligomer (i.e. duplexes)—in this regard, the oligomer is not(essentially) double stranded. In some embodiments, the oligomer isessentially not double stranded. In some embodiments, the oligomer isnot a siRNA. In various embodiments, the oligomer of the invention canconsist entirely of the contiguous nucleotide region. Thus, in someembodiments the oligomer is not substantially self-complementary.

In other embodiments, the present invention includes fragments ofoligomers. For example, the invention includes at least one nucleotide,at least two contiguous nucleotides, at least three contiguousnucleotides, at least four contiguous nucleotides, at least fivecontiguous nucleotides, at least six contiguous nucleotides, at leastseven contiguous nucleotides, at least eight contiguous nucleotides, orat least nine contiguous nucleotides of the oligomers disclosed herein.Fragments of any of the sequences disclosed herein are contemplated aspart of the invention.

II.A. The Target

Suitably the oligomer of the invention is capable of down-regulating(e.g., reducing or removing) expression of the MAPT mRNA or protein. Inthis regard, the oligomer of the invention can affect indirectinhibition of Tau protein through the reduction in Tau mRNA levels,typically in a mammalian cell, such as a human cell, such as a neuronalcell.

Microtubule-associated protein tau (MAPT), in a pathologic stateassociated with disease, is also known as neurofibrillary tangle proteinor paired helical filament-tau (PHF-tau). Synonyms of MAPT are known andinclude DDPAC, FTDP-17L, MSTD, MTBT1, MTBT2, PPND, PPP1R103, MAPTL, andTAU. The sequence for the MAPT gene can be found under publiclyavailable Accession Number NC_000017.11 and the sequence for the MAPTpre-mRNA transcript can be found under publicly available AccessionNumber NG 007398 (SEQ ID NO: 1). The sequence for Tau protein can befound under publicly available Accession Numbers: P10636, P18518,Q14799, Q15549, Q15550, Q15551, Q1RMF6, Q53YB1, Q5CZI7, Q5XWFO, Q6QT54,Q9UDJ3, Q9UMHO, Q9UQ96, each of which is incorporated by referenceherein in its entirety. Natural variants of the MAPT gene product areknown. For example, natural variants of Tau protein can contain one ormore amino acid substitutions selected from: R5H, R5L, D285N, V289A,K574T, L583V, G589V, N596K, N613H, P618L, P618S, G620V, S622N, K634M,S637F, V654M, E659V, K6861, G706R, R723W, and any combinations thereof.Therefore, the oligomers of the present invention can be designed toreduce or inhibit expression of the natural variants of the Tau protein.

Mutations in Tau are known to cause one or more pathological conditions.The oligomers of the invention can be used to reduce or inhibit theexpression of a SNP or alternatively spliced MAPT transcript containingone or more mutations and consequently reduce the formation of a mutatedTau protein. Examples of Tau protein mutants include, but are notlimited to a Tau protein comprising one or more mutations selected from:S515E, S516E, S519E, S531A, T548A, T548E, S552A, S552E, S579A, S713E,S721E, S726E, S730E, S739E, and any combination thereof. The oligomer ofthe invention can be designed to reduce or inhibit expression of anymutants of Tau proteins.

An example of a target nucleic acid sequence of the oligomers is MAPTpre-mRNA or MAPT mRNA. SEQ ID NO: 1 in FIG. 1A represents a MAPT genomicsequence. SEQ ID NO: 1 is identical to a MAPT pre-mRNA sequence exceptthat nucleotide “t” in SEQ ID NO: 1 is shown as “u” in pre-mRNA. SEQ IDNO: 2 in FIG. 1B represents a MAPT mRNA sequence except that nucleotide1 in SEQ ID NO: 2 is shown as “u” in mRNA. In certain embodiments, the“target nucleic acid” comprises a Tau protein-encoding nucleic acids ornaturally occurring variants thereof, and RNA nucleic acids derivedtherefrom, e.g., pre-mRNA or mature mRNA. In some embodiments, forexample when used in research or diagnostics the “target nucleic acid”can be a cDNA or a synthetic oligonucleotide derived from the above DNAor RNA nucleic acid targets. In one embodiment, the MAPT genomicsequence is shown as GenBank Accession No. NG 007398.1 (SEQ ID NO: 1).The 3′ UTR region of the MAPT pre-mRNA is known to correspond tonucleotides 134,947-140,924 of SEQ ID NO: 1. The 5′ UTR region of theMAPT pre-mRNA is known to correspond to nucleotides 1-72,917 of SEQ IDNO: 1. MAPT cDNA which corresponds to MAPT mRNA is known as GenBankAccession No. NM_016835.3 (SEQ ID NO: 2). See FIG. 1B. The Tau proteinsequence encoded by the MAPT mRNA is shown as SEQ ID NO: 3. See FIG. 1C.

In some embodiments, an oligomer of the invention hybridizes to a regionwithin the 3′ UTR of a MAPT transcript, e.g., SEQ ID NO: 1. In someembodiments, an oligomer of the invention hybridizes to a region withinthe 3′ UTR of a MAPT transcript, e.g., SEQ ID NO: 1, wherein theoligomer has a design according to formula: 5′ A-B-C 3′ as describedelsewhere herein (e.g., Section II.G, e.g., Section II.G.I) or achemical structure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B).In certain embodiments, the oligomers hybridize to a region within a 3′UTR of a MAPT transcript, e.g., SEQ ID NO: 1, and have a sequence scoreequal to or greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, or 1.0. Calculation methods of the sequence score are disclosedelsewhere herein.

In one embodiment, the oligomer according to the invention comprises acontiguous nucleotide sequence that hybridizes to a region within 3′ UTRin a microtubule-associated protein MAPT transcript, e.g., a regioncorresponding to the 3′ UTR of SEQ ID NO: 1. In another embodiment, theoligomer of the invention comprises a contiguous nucleotide sequencethat hybridizes to a nucleic acid sequence, or a region within thesequence, of a MAPT transcript (“target region”), wherein the nucleicacid sequence corresponds to nucleotides 134,947-138,940 of SEQ IDNO: 1. In another embodiment, the oligomer of the invention comprises acontiguous nucleotide sequence that hybridizes to a nucleic acidsequence, or a region within the sequence, of a MAPT transcript, whereinthe nucleic acid sequence corresponds to nucleotides 134,947-138,940 ofSEQ ID NO: 1, and wherein the oligomer has one of the designs describedherein (e.g., Section II.G. e.g., a gapmer design, e.g., an alternatingflank gapmer design) or a chemical structure shown elsewhere herein(e.g., FIGS. 2, 16B, and 20B). In another embodiment, the target regioncorresponds to nucleotides 134,947-138,924 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides135,050-138,940; 135,700-138,940; 136,000-138,940; 136,620-138,940;136,860-138,940; 137,060-138,940; 137,300-138,940; 137,830-138,940;138,030-138,940; 138,350-138,940; 134,821-135,020; 135,050-135,820;135,700-135,820; 136,000-136,110; 136,010-136,100; 136,020-136,090;136,030-136,080; 136,040-136,070; 136,620-136,760; 136,860-136,960;137,060-137,110; 137,300-137,400; 137,830-137,900; 138,030-138,140;138,350-138,450; 138,860-138,940; 138,870-138,930; 138,880-138,920; or138,890-138,920 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G.I, e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In another embodiment,the target region corresponds to nucleotides 135,050-138,940 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 135,700-138,940 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 136,000-138,940 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 136,620-138,940 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 136,860-138,940 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 137,060-138,940 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 137,300-138,940 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 137,830-138,940 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 138,030-138,940 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 138,350-138,940 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 134,821-135,020 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 135,050-138,820 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 135,700-135,820 of SEQ IDNO: 1. In other embodiments, the target region corresponds tonucleotides 136,620-136,760 of SEQ ID NO: 1. In some embodiments, thetarget region corresponds to nucleotides 136,860-136,960 of SEQ IDNO: 1. In certain embodiments, the target region corresponds tonucleotides 137,060-137,110 of SEQ ID NO: 1. In other embodiments, thetarget region corresponds to nucleotides 137,300-137,400 of SEQ IDNO: 1. In yet other embodiments, the target region corresponds tonucleotides 137,830-137,900 of SEQ ID NO: 1. In still other embodiments,the target region corresponds to nucleotides 138,030-138,140 of SEQ IDNO: 1. In certain embodiments, the target region corresponds tonucleotides 138,350-138,450 of SEQ ID NO: 1. In other embodiments, thetarget region corresponds to nucleotides 138,860-138,940 of SEQ ID NO:1.

In some embodiments, the target region corresponds to nucleotides134,947-134,989, 135,533-135,550, 135,585-135,605, 135,690-135,710,135,739-135,769, 135,775-135,792, 136,049-136,070, 136,053-136,068;136,650-136,667, 136,693-136,723, 136,896-136,926, 137,067-137,089,137,326-137,373, 137,851-137,883, 138,058-138,119, 138,377-138,394,138,401-138,420, 138,884-138,908; 138,401-138,908; 138,377-138,908;138,058-138,908; 137,851-138,908; 137,326-138,908; 137,067-138,908;136,896-138,908; 136,693-138,908; 136,650-138,908; 136,049-138,908;135,775-138,908; 135,739-138,908; or 134,947-138,908 of SEQ ID NO: 1,wherein optionally, the oligomer has a design described elsewhere herein(e.g., Section II.G, e.g., a gapmer design, e.g., an alternating flankgapmer design). In another embodiment, the target region corresponds tonucleotides 134,947-134,989 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 135,533-135,550 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 135,585-135,605 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 135,690-135,710 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 135,739-135,769 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 135,775-135,792 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 136,049-136,070 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 136,053-136,068 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 136,650-136,667 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 136,693-136,723 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 136,896-136,926 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 137,067-137,089 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 137,326-137,373 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 137,851-137,883 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 138,058-138,119 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 138,377-138,394 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 138,401-138,420 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 138,884-138,908 of SEQ ID NO:1.

In other embodiments, the target region corresponds to nucleotides138,401-138,908 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 138,377-138,908 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides138,058-138,908 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 137,851-138,908 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides137,326-138,908 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 137,067-138,908 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides136,896-138,908 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 136,693-138,908 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides136,650-138,908 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 136,049-138,908 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides135,775-138,908 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 135,739-138,908 of SEQ ID NO: 1.

In other embodiments, the target region corresponds to nucleotides136,053-136,068 of SEQ ID NO: 1+1, +2, +3, +4, +5, +6, +7, +8, +9, +10,+11, +12, +13, +14, +15, +20, +25, +30, +35, +40, +45, or +50nucleotides at the 3′ end, the 5′ end, or both. In certain embodiments,the target region corresponds to nucleotides 138,884-138,908 of SEQ IDNO: 1+1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15,+20, +25, +30, +35, +40, +45, or +50 nucleotides of SEQ ID NO: 1 at the3′ end, the 5′ end, or both.

In some embodiments, an oligomer of the invention hybridizes to a regionwithin the 5′ UTR of a MAPT transcript, e.g., SEQ ID NO: 1. In someembodiments, an oligomer of the invention hybridizes to a region withinthe 5′ UTR of a MAPT transcript, e.g., SEQ ID NO: 1, wherein theoligomer has a design according to formula: 5′ A-B-C 3′ as describedelsewhere herein (e.g., Section II.G, e.g., Section II.G.I) or achemical structure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B).In certain embodiments, the oligomers hybridize to a region within a 5′UTR of a MAPT transcript, e.g., SEQ ID NO: 1, and have a sequence scoreequal to or greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, or 1.0. Calculation methods of the sequence score are disclosedelsewhere herein.

In some embodiments, an oligomer of the invention hybridizes to a regionwithin exon 2 of a MAPT transcript, e.g., SEQ ID NO: 1. In someembodiments, an oligomer of the invention hybridizes to a region withinexon 2 of a MAPT transcript, e.g., SEQ ID NO: 1, wherein the oligomerhas a design according to formula: 5′ A-B-C 3′ as described elsewhereherein (e.g., Section II.G, e.g., Section II.G.I) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, the oligomers hybridize to a region within exon 2of a MAPT transcript, e.g., SEQ ID NO: 1, and have a sequence scoreequal to or greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, or 1.0. Calculation methods of the sequence score are disclosedelsewhere herein.

In one embodiment, the oligomer according to the invention comprises acontiguous nucleotide sequence that hybridizes to a region within 5′ UTRand/or exon 2 in a microtubule-associated protein MAPT transcript, e.g.,a region corresponding to the 5′ UTR and/or exon 2 of SEQ ID NO: 1. Inthe MAPT transcript, the 5′ UTR and exon 2 overlap but are notcontiguous. In another embodiment, the oligomer of the inventioncomprises a contiguous nucleotide sequence that hybridizes to a nucleicacid sequence, or a region within the sequence, of a MAPT transcript(“target region”), wherein the nucleic acid sequence corresponds tonucleotides 72,802-73,072 of SEQ ID NO: 1. In another embodiment, theoligomer of the invention comprises a contiguous nucleotide sequencethat hybridizes to a nucleic acid sequence, or a region within thesequence, of a MAPT transcript (“target region”), wherein the nucleicacid sequence corresponds to nucleotides 72,802-73,072 of SEQ ID NO: 1,and wherein the oligomer has one of the designs described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In another embodiment, the target region corresponds tonucleotides 72,802-73,072; 72,812-73,062; 72,822-73,052; 72,832-73,042;72,842-73,032; 72,852-73,022; 72,862-73,012; 72,872-73,002;72,882-72,992; 72,892-72,982; or 72,902-72,972 of SEQ ID NO: 1, whereinoptionally, the oligomer has a design described herein (e.g., SectionII.G.I, e.g., a gapmer design, e.g., an alternating flank gapmer design)or a chemical structure shown elsewhere herein (e.g., FIGS. 2, 16B, and20B). In another embodiment, the target region corresponds tonucleotides 72,802-73,072; 72 of SEQ ID NO: 1. In another embodiment,the target region corresponds to nucleotides 72,812-73,062 of SEQ IDNO: 1. In another embodiment, the target region corresponds tonucleotides 72,822-73,052 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,832-73,042 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,842-73,032 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,852-73,022 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,862-73,012of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,872-73,002 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,882-72,992 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,892-72,982 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,902-72,972 of SEQ ID NO: 1.

In some embodiments, the target region corresponds to nucleotides72,802-73,072; 72,812-73,072; 72,822-73,072; 72,832-73,072;72,842-73,072; 72,852-73,072; 72,862-73,072; 72,872-73,072;72,882-73,072; 72,892-73,072; 72,902-73,072; 72,802-73,062;72,802-73,052; 72,802-73,042; 72,802-73,032; 72,802-73,022;72,802-73,012; 72,802-73,002; 72,802-72,992; 72,802-73,982; or72,802-73,972 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described elsewhere herein (e.g., Section II.G, e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Inanother embodiment, the target region corresponds to nucleotides72,802-73,072 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,812-73,072 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,822-73,072of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,832-73,072 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,842-73,072 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,852-73,072 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,862-73,072 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,872-73,072of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,882-73,072 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,892-73,072 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,902-73,072 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,802-73,062 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,802-73,052of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,802-73,042 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,802-73,032 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,802-73,022 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,802-73,012 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,802-73,002of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,802-72,992 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,802-73,982 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,802-73,972 of SEQ ID NO: 1.

In some embodiments, the target region corresponds to nucleotides72,862-73,012; 72,872-73,012; 72,882-73,012; 72,892-73,012;72,902-73,012; 72,862-73,002; 72,872-73,002; 72,882-73,002;72,892-73,002; 72,902-73,002; 72,862-72,992; 72,872-72,992;72,882-72,992; 72,892-72,992; 72,902-72,992; 72,862-72,982;72,872-72,982; 72,882-72,982; 72,892-72,982; 72,902-72,982;72,862-72,972; 72,872-72,972; 72,882-72,972; 72,892-72,972; or72,902-72,972 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described elsewhere herein (e.g., Section II.G, e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Inanother embodiment, the target region corresponds to nucleotides72,872-73,012 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,882-73,012 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,892-73,012of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,902-73,012 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,862-73,002 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,872-73,002 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,882-73,002 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,892-73,002of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,902-73,002 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,862-72,992 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,872-72,992 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,882-72,992 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,892-72,992of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,902-72,992 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,862-72,982 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,872-72,982 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,882-72,982 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,892-72,982of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,902-72,982 of SEQ ID NO: 1. In another embodiment, thetarget region corresponds to nucleotides 72,862-72,972 of SEQ ID NO: 1.In another embodiment, the target region corresponds to nucleotides72,872-72,972 of SEQ ID NO: 1. In another embodiment, the target regioncorresponds to nucleotides 72,882-72,972 of SEQ ID NO: 1. In anotherembodiment, the target region corresponds to nucleotides 72,892-72,972of SEQ ID NO: 1. In another embodiment, the target region corresponds tonucleotides 72,902-72,972 of SEQ ID NO: 1.

In other embodiments, the target region corresponds to nucleotides72,947-72,960 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 72,946-72,961 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides 72,907-72,922of SEQ ID NO: 1. In other embodiments, the target region corresponds tonucleotides 72,948-72,963 of SEQ ID NO: 1. In other embodiments, thetarget region corresponds to nucleotides 72,950-72,963 of SEQ ID NO: 1.In other embodiments, the target region corresponds to nucleotides72,945-72,960 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 72,950-72,965 of SEQ ID NO: 1.

In other embodiments, the target region corresponds to nucleotides72,944-72,959 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 72,947-72,962 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides 72,952-72,965of SEQ ID NO: 1. In other embodiments, the target region corresponds tonucleotides 72,946-72,959 of SEQ ID NO: 1. In other embodiments, thetarget region corresponds to nucleotides 72,949-72,964 of SEQ ID NO: 1.In other embodiments, the target region corresponds to nucleotides72,951-72,964 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 72,933-72,948 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides 72,934-72,949of SEQ ID NO: 1. In other embodiments, the target region corresponds tonucleotides 72,935-72,950 of SEQ ID NO: 1. In other embodiments, thetarget region corresponds to nucleotides 72,932-72,951 of SEQ ID NO: 1.In other embodiments, the target region corresponds to nucleotides72,933-72,952 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 72,934-72,953 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides 72,935-72,954of SEQ ID NO: 1.

In other embodiments, the target region corresponds to nucleotides72,944-72,963 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 72,945-72,964 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides 72,946-72,965of SEQ ID NO: 1.

In other embodiments, the target region corresponds to nucleotides72,948-72,967 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 72,933-72,949 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides 72,935-72,951of SEQ ID NO: 1. In other embodiments, the target region corresponds tonucleotides 72,936-72,953 of SEQ ID NO: 1. In other embodiments, thetarget region corresponds to nucleotides 72,933-72,934 of SEQ ID NO: 1.In other embodiments, the target region corresponds to nucleotides72,934-72,950 of SEQ ID NO: 1. In other embodiments, the target regioncorresponds to nucleotides 72,934-72,951 of SEQ ID NO: 1. In otherembodiments, the target region corresponds to nucleotides 72,933-72,954of SEQ ID NO: 1. In other embodiments, the target region corresponds tonucleotides 72,933-72,950 of SEQ ID NO: 1.

In other embodiments, an oligomer of the invention comprises acontiguous nucleotide sequence that hybridizes to a nucleic acidsequence, or a region within the sequence, of a MAPT transcript (“targetregion”), wherein the nucleic acid sequence corresponds to nucleotides97,648-97,661 of SEQ ID NO: 1, and wherein the oligomer optionally hasone of the designs described herein (e.g., Section II.G. e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B).

In yet other embodiments, an oligomer of the invention comprises acontiguous nucleotide sequence that hybridizes to a nucleic acidsequence, or a region within the sequence, of a MAPT transcript (“targetregion”), wherein the nucleic acid sequence corresponds to nucleotides134,749-134,764 of SEQ ID NO: 1, and wherein the oligomer optionally hasone of the designs described herein (e.g., Section II.G. e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B).

In certain embodiments, the oligomer of the invention is capable ofhybridizing to the target nucleic acid (e.g., MAPT transcript) underphysiological condition, i.e., in vivo condition. In some embodiments,the oligomer of the invention is capable of hybridizing to the targetnucleic acid (e.g., MAPT transcript) in vitro. In some embodiments, theoligomer of the invention is capable of hybridizing to the targetnucleic acid (e.g., MAPT transcript) in vitro under stringentconditions. Stringency conditions for hybridization in vitro aredependent on, inter alia, productive cell uptake, RNA accessibility,temperature, free energy of association, salt concentration, and time(see, e.g., Stanley T Crooks, Antisense Drug Technology: Principles,Strategies and Applications, 2^(nd) Edition, CRC Press (2007))).Generally, conditions of high to moderate stringency are used for invitro hybridization to enable hybridization between substantiallysimilar nucleic acids, but not between dissimilar nucleic acids. Anexample of stringent hybridization conditions include hybridization in5× saline-sodium citrate (SSC) buffer (0.75 M sodium chloride/0.075 Msodium citrate) for 1 hour at 40° C., followed by washing the sample 10times in 1×SSC at 40° C. and 5 times in 1×SSC buffer at roomtemperature. In vivo hybridization conditions consist of intracellularconditions (e.g., physiological pH and intracellular ionic conditions)that govern the hybridization of antisense oligonucleotides with targetsequences. In vivo conditions can be mimicked in vitro by relatively lowstringency conditions. For example, hybridization can be carried out invitro in 2×SSC (0.3 M sodium chloride/0.03 M sodium citrate), 0.1% SDSat 37° C. A wash solution containing 4×SSC, 0.1% SDS can be used at 37°C., with a final wash in 1×SSC at 45° C.

II.B. Oligomer Sequences

The oligomers of the invention comprise a contiguous nucleotide sequencewhich corresponds to the complement of a region of MAPT transcript,e.g., a nucleotide sequence corresponding to SEQ ID NO: 1.

In certain embodiments, the invention provides an oligomer from 10-50,such as 10-30 nucleotides in length which comprises a contiguousnucleotide sequence of a total of from 10-30 nucleotides, wherein thecontiguous nucleotide sequence has at least 85%, 90%, 95%, 98%, or 99%)sequence identity to a region within the complement of a mammalianmicrotubule-associated protein tau (MAPT) transcript, such as SEQ ID NO:1 or naturally occurring variant thereof. Thus, for example, theoligomer hybridizes to a single stranded nucleic acid molecule havingthe sequence of a portion of SEQ ID NO: 1.

The oligomer can comprise a contiguous nucleotide sequence which isfully complementary (perfectly complementary) to the equivalent regionof a nucleic acid which encodes a mammalian Tau protein (e.g., SEQ IDNO: 1). The oligomer can comprise a contiguous nucleotide sequence whichis fully complementary (perfectly complementary) to a nucleic acidsequence, or a region within the sequence, corresponding to nucleotides134,947-138,940, 135,050-138,940; 135,700-138,940; 136,000-138,940;136,620-138,940; 136,860-138,940; 137,060-138,940; 137,300-138,940;137,830-138,940; 138,030-138,940; 138,350-138,940; 134,821-135,020;135,050-135,820; 135,700-135,820; 136,000-136,110; 136,620-136,760;136,860-136,960; 137,060-137,110; 137,300-137,400; 137,830-137,900;138,030-138,140; 138,350-138,450; or 138,860-138,940 of SEQ ID NO: 1.Furthermore, the oligomer can have a design described elsewhere herein(e.g., Section II.G. e.g., a gapmer design, e.g., an alternating flankgapmer design) or a chemical structure shown elsewhere herein (e.g.,FIGS. 2, 16B, and 20B).

The oligomer can also comprise a contiguous nucleotide sequence which isfully complementary (perfectly complementary) to the equivalent regionof a nucleic acid which encodes a mammalian Tau protein (e.g., SEQ IDNO: 1). The oligomer can comprise a contiguous nucleotide sequence whichis fully complementary (perfectly complementary) to a nucleic acidsequence, or a region within the sequence, corresponding to nucleotides72,862-73,012; 72,872-73,012; 72,882-73,012; 72,892-73,012;72,902-73,012; 72,862-73,002; 72,872-73,002; 72,882-73,002;72,892-73,002; 72,902-73,002; 72,862-72,992; 72,872-72,992;72,882-72,992; 72,892-72,992; 72,902-72,992; 72,862-72,982;72,872-72,982; 72,882-72,982; 72,892-72,982; 72,902-72,982;72,862-72,972; 72,872-72,972; 72,882-72,972; 72,892-72,972; or72,902-72,972 of SEQ ID NO: 1. Furthermore, the oligomer can have adesign described elsewhere herein (e.g., Section II.G. e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B).

In certain embodiments, the nucleotide sequence of the oligomers of theinvention or the contiguous nucleotide sequence has at least about 80%sequence identity to a sequence selected from SEQ ID NOs: 4 to 803, and901 to 935 (i.e., the sequences in FIGS. 2, 3, 6, and 7), such as atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96% sequence identity, at least 97%sequence identity, at least 98% sequence identity, at least 99% sequenceidentity, such as 100% sequence identity (homologous). In someembodiments, the oligomer has a design described elsewhere herein (e.g.,Section II.G.I, e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B).

In other embodiments, the oligomer of the invention comprises at leastone oligomer sequence (e.g., ASO number) disclosed in FIGS. 2, 3, 6, 7,16A, 16B, 20A or 20B. In some embodiments, the oligomer of the inventioncomprises at least one oligomer sequence (e.g., ASO number) disclosed inFIGS. 2, 3, 6, 7, 16A, 16B, 20A or 20B wherein the oligomer is onenucleotide, two nucleotides, three nucleotides, four nucleotides, fivenucleotides, six nucleotides, seven nucleotides, eight nucleotides, ninenucleotides, ten nucleotides, 11 nucleotides, 12 nucleotides, 13nucleotides, 14 nucleotides, or 15 nucleotides shorter at the 3′ endthan the ASOs disclosed in FIGS. 2, 3, 6, 7, 16A, 16B, 20A or 20B. Inother embodiments, the oligomer of the invention comprises at least oneoligomer sequence (e.g., ASO number) disclosed in FIGS. 2, 3, 6, 7, 16A,16B, 20A or 20B, wherein the oligomer is one nucleotide, twonucleotides, three nucleotides, four nucleotides, five nucleotides, sixnucleotides, seven nucleotides, eight nucleotides, nine nucleotides, tennucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14nucleotides, or 15 nucleotides shorter at the 5′ end than the ASOsdisclosed in FIGS. 2, 3, 6, 7, 16A, 16B, 20A or 20B. In yet otherembodiments, the oligomer of the invention comprises at least oneoligomer sequence (e.g., ASO number) disclosed in FIGS. 2, 3, 6, 7, 16A,16B, 20A or 20B, wherein the oligomer is one nucleotide, twonucleotides, three nucleotides, four nucleotides, five nucleotides, sixnucleotides, seven nucleotides, eight nucleotides, nine nucleotides, tennucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14nucleotides, or 15 nucleotides shorter at the 5′ end and/or the 3′ endthan the ASOs disclosed in FIGS. 2, 3, 6, 7, 16A, 16B, 20A or 20B.

In certain embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of a nucleic acidsequence selected from nucleotides 134,947-134,989, 135,739-135,769,135,775-135,792, 136,049-136,070, 136,053-136,068; 136,650-136,667,136,693-136,723, 136,896-136,926, 137,067-137,089, 137,326-137,373,137,851-137,883, 138,058-138,119, 138,377-138,394, 138,401-138,420,138,884-138,924; 138,401-138,924; 138,377-138,924; 138,058-138,924;137,851-138,924; 137,326-138,924; 137,067-138,924; 136,896-138,924;136,693-138,924; 136,650-138,924; 136,049-138,924; 135,775-138,924;135,739-138,924; 134,947-138,924; 134,947-138,940; 134,909-138,924;134,871-138,924; and 134,854-138,924 of SEQ ID NO: 1. In someembodiments, the oligomer has a design described elsewhere herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B).

In certain embodiments, the nucleotide sequence of the oligomers of theinvention or the contiguous nucleotide sequence has at least about 80%sequence identity to a sequence selected from SEQ ID NOs: 804 to 900(i.e., the sequences in FIG. 16A or 16B), such as at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96% sequence identity, at least 97% sequence identity, atleast 98% sequence identity, at least 99% sequence identity, such as100% sequence identity (homologous). In some embodiments, the oligomerhas a design described elsewhere herein (e.g., Section II.G.I, e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B).

In certain embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of a nucleic acidsequence selected from nucleotides 72,862-73,012; 72,872-73,012;72,882-73,012; 72,892-73,012; 72,902-73,012; 72,862-73,002;72,872-73,002; 72,882-73,002; 72,892-73,002; 72,902-73,002;72,862-72,992; 72,872-72,992; 72,882-72,992; 72,892-72,992;72,902-72,992; 72,862-72,982; 72,872-72,982; 72,882-72,982;72,892-72,982; 72,902-72,982; 72,862-72,972; 72,872-72,972;72,882-72,972; 72,892-72,972; and 72,902-72,972 of SEQ ID NO: 1. In someembodiments, the oligomer has a design described elsewhere herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B).

In certain embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides134,947-138,940 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 135,700-138,940 of SEQ IDNO: 1, wherein optionally, the oligomer has a design described herein(e.g., Section II.G. e.g., a gapmer design, e.g., an alternating flankgapmer design) or a chemical structure shown elsewhere herein (e.g.,FIGS. 2, 16B, and 20B).

In certain embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides136,000-138,940 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 136,620-138,940 of SEQ IDNO: 1, wherein optionally, the oligomer has a design described herein(e.g., Section II.G. e.g., a gapmer design, e.g., an alternating flankgapmer design) or a chemical structure shown elsewhere herein (e.g.,FIGS. 2, 16B, and 20B).

In certain embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides136,860-138,940 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 137,060-138,940 of SEQ IDNO: 1, wherein optionally, the oligomer has a design described herein(e.g., Section II.G. e.g., a gapmer design, e.g., an alternating flankgapmer design) or a chemical structure shown elsewhere herein (e.g.,FIGS. 2, 16B, and 20B). In certain embodiments, an oligomer of theinvention comprises a nucleotide sequence having at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99%, or 100% sequence identity to a region within thecomplement of nucleotides 137,300-138,940 of SEQ ID NO: 1, whereinoptionally, the oligomer has a design described herein (e.g., SectionII.G. e.g., a gapmer design, e.g., an alternating flank gapmer design)or a chemical structure shown elsewhere herein (e.g., FIGS. 2, 16B, and20B). In certain embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides137,830-138,940 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 138,030-138,940 of SEQ IDNO: 1, wherein optionally, the oligomer has a design described elsewhereherein (e.g., Section II.G. e.g., a gapmer design, e.g., an alternatingflank gapmer design) or a chemical structure shown elsewhere herein(e.g., FIGS. 2, 16B, and 20B). In certain embodiments, an oligomer ofthe invention comprises a nucleotide sequence having at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or 100% sequence identity to a region within thecomplement of nucleotides 138,350-138,940 of SEQ ID NO: 1, whereinoptionally, the oligomer has a design described elsewhere herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 136,000-136,110 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described elsewhere herein (e.g., Section II.G.e.g., a gapmer design, e.g., an alternating flank gapmer design) or achemical structure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B).

In other embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides138,860-138,940 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described elsewhere herein (e.g., Section II.G. e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides138,884-138,908 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described elsewhere herein (e.g., Section II.G. e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). In otherembodiments, an oligomer of the invention has at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99%, or 100% sequence identity to a region within thecomplement of nucleotides 134,854-138,924 of SEQ ID NO: 1, whereinoptionally, the oligomer has a design described elsewhere herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In some embodiments, an oligomer of the invention has atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of a nucleic acid sequence selected fromnucleotides 134,947-134,989, 135,739-135,769, 135,775-135,792,136,049-136,070, 136,053-136,068; 136,650-136,667, 136,693-136,723,136,896-136,926, 137,067-137,089, 137,326-137,373, 137,851-137,883,138,058-138,119, 138,377-138,394, 138,401-138,420, 138,884-138,924;138,401-138,924; 138,377-138,924; 138,058-138,924; 137,851-138,924;137,326-138,924; 137,067-138,924; 136,896-138,924; 136,693-138,924;136,650-138,924; 136,049-138,924; 135,775-138,924; 135,739-138,924; and134,947-138,924 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described elsewhere herein (e.g., Section II.G. e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). In someembodiments, the region is within the complement of nucleotides134,947-134,989 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 135,739-135,769 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides135,775-135,792 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 136,049-136,070 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides136,053-136,068 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 136,650-136,667 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides136,693-136,723 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 136,896-136,926 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides137,067-137,089 of SEQ ID NO: 1.

In other embodiments, the region is within the complement of nucleotides137,326-137,373 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 137,851-137,883 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides138,058-138,119 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 138,377-138,394 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides138,401-138,420 of SEQ ID NO: 1.

In some embodiments, the region is within the complement of nucleotides138,884-138,924 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 138,401-138,924 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides138,377-138,924 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 138,058-138,924 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides137,851-138,924 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 137,326-138,924 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides137,067-138,924 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 136,896-138,924 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides136,693-138,924 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 136,650-138,924 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides136,049-138,924 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 135,775-138,924 of SEQ ID NO: 1. Insome embodiments, the region is within the complement of nucleotides135,739-138,924 of SEQ ID NO: 1. In some embodiments, the region iswithin the complement of nucleotides 134,947-138,924 of SEQ ID NO: 1.

In some embodiments the oligomer (or contiguous nucleotide portionthereof) is selected from, or comprises, one of the sequences selectedfrom the group consisting of SEQ ID NOs: 4 to 803, 901 to 953, 956 to958, and 960 or a region of at least 10 contiguous nucleotides thereof,wherein the oligomer (or contiguous nucleotide portion thereof) canoptionally comprise one, two, three, or four mismatches when compared tothe corresponding MAPT transcript.

In one embodiment, the oligomer can comprise a sequence selected fromthe group consisting of ctttatttccaaattcactt [138888-138907] (SEQ ID NO:676); actttatttccaaattcact [138889-138908] (SEQ ID NO: 715);tttatttccaaattcacttt [138887-138906] (SEQ ID NO: 644);ttatttccaaattcactttt [138886-138905] (SEQ ID NO: 799);atttccaaattcacttttac [138884-138903](SEQ ID NO: 466);atttccaaattcactttta [138885-138903] (SEQ ID NO: 559);actttatttccaaattcactt [138888-138908] (SEQ ID NO: 680); atttccaaattcactt[138888-138903] (SEQ ID NO: 686); tatttccaaattcactttta [13885-138904](SEQ ID NO: 526); aataactttatttcca [138897-138912] (SEQ ID NO: 773);agtaataactttatt [138901-138915] (SEQ ID NO: 782); tttccaaattcactt[138888-138902] (SEQ ID NO: 684); agagtaataactttat [138902-138917] (SEQID NO: 784); agtaataactttattt [138900-138915] (SEQ ID NO: 780);agagtaataacttta [138903-138917] (SEQ ID NO: 786); ttaatcagagtaataa[138908-138923] (SEQ ID NO: 795); tttaatcagagtaat [138910-138924] (SEQID NO: 798); aatcagagtaataac [138907-138921] (SEQ ID NO: 794);tttaatcagagtaata [138909-139924] (SEQ ID NO: 797);taatcagagtaataa[138908-138922] (SEQ ID NO: 796); ctttatttccaaattcact[138889-138907] (SEQ ID NO: 713); or ctttatttccaaattcac [138890-138907](SEQ ID NO: 739). In a particular embodiment, the oligomer comprisesatttccaaattcacttttac [138884-138903] (SEQ ID NO: 466). In oneembodiment, the oligomer (or contiguous nucleotide portion thereof)optionally has one, two, three, or four mismatches against the selectedsequence. In another embodiment, the oligomer optionally comprises oneor more nucleotide analogs. In other embodiments, the oligomer has adesign described elsewhere herein (e.g., Section II.G.I, e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B).Non-limiting examples of the nucleotide analogs useful for the inventionare disclosed elsewhere herein.

In other embodiments, an oligomer of the invention comprises, consistsessentially of, or consists of a nucleotide sequence having at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or 100% sequence identity to SEQ ID NOs:939, 940, 524, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 956,957, 958, 951, 952, or 953. In some embodiments, the oligomer having asequence identity to SEQ ID NOs: 939, 940, 524, 941, 942, 943, 944, 945,946, 947, 948, 949, 950, 956, 957, 958, 951, 952, or 953 has the designof ASO-257283, ASO-257284, ASO-002263, ASO-002627, ASO-002677,ASO-002670, ASO-002663, ASO-002635, ASO-002643, ASO-002671, ASO-002664,ASO-002626, ASO-002634, ASO-002678, ASO-002650, ASO-002657, ASO-002642,ASO-002649, or ASO-002656, respectively.

In certain embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,802-73,072 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,812-73,062 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,822-73,052 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement nucleotides72,832-73,042 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,842-73,032 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,852-73,022 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,862-73,012 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B).

In certain embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,872-73,002 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,882-72,992 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,892-72,982 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,902-72,972 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,802-73,072 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,812-73,072 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,822-73,072 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,832-73,072 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,842-73,072 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B).

In certain embodiments, an oligomer of the invention comprises anucleotide sequence having at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,852-73,072 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,862-73,072 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,872-73,072 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,882-73,072 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,892-73,072 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,902-73,072 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,802-73,062 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,802-73,052 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,802-73,042 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,802-73,032 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,802-73,022 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,802-73,012 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,802-73,002 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B). In certain embodiments,an oligomer of the invention comprises a nucleotide sequence having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to aregion within the complement of nucleotides 72,802-72,992 of SEQ ID NO:1, wherein optionally, the oligomer has a design described herein (e.g.,Section II.G. e.g., a gapmer design, e.g., an alternating flank gapmerdesign) or a chemical structure shown elsewhere herein (e.g., FIGS. 2,16B, and 20B). In certain embodiments, an oligomer of the inventioncomprises a nucleotide sequence having at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or 100% sequence identity to a region within the complementof nucleotides 72,802-73,982 of SEQ ID NO: 1, wherein optionally, theoligomer has a design described herein (e.g., Section II.G. e.g., agapmer design, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). Incertain embodiments, an oligomer of the invention comprises a nucleotidesequence having at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a region within the complement of nucleotides72,802-73,972 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described herein (e.g., Section II.G. e.g., a gapmer design,e.g., an alternating flank gapmer design) or a chemical structure shownelsewhere herein (e.g., FIGS. 2, 16B, and 20B).

In some embodiments, an oligomer of the invention has at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% sequence identity to a region withinthe complement of a nucleic acid sequence selected from nucleotides72,862-73,012; 72,872-73,012; 72,882-73,012; 72,892-73,012;72,902-73,012; 72,862-73,002; 72,872-73,002; 72,882-73,002;72,892-73,002; 72,902-73,002; 72,862-72,992; 72,872-72,992;72,882-72,992; 72,892-72,992; 72,902-72,992; 72,862-72,982;72,872-72,982; 72,882-72,982; 72,892-72,982; 72,902-72,982;72,862-72,972; 72,872-72,972; 72,882-72,972; 72,892-72,972; and72,902-72,972 of SEQ ID NO: 1, wherein optionally, the oligomer has adesign described elsewhere herein (e.g., Section II.G. e.g., a gapmerdesign, e.g., an alternating flank gapmer design) or a chemicalstructure shown elsewhere herein (e.g., FIGS. 2, 16B, and 20B). In someembodiments, the region is within the complement of nucleotides72,862-73,012 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,872-73,012 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,882-73,012 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,892-73,012 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,902-73,012 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,862-73,002 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,872-73,002 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,882-73,002 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,892-73,002 of SEQ ID NO: 1.

In other embodiments, the region is within the complement of nucleotides72,902-73,002 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,862-72,992 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,872-72,992 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,882-72,992 of SEQ ID NO: 1.

In some embodiments, the region is within the complement of nucleotides72,892-72,992 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,902-72,992 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,862-72,982 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,872-72,982 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,882-72,982 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,892-72,982 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,902-72,982 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,862-72,972 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,872-72,972 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,882-72,972 of SEQ ID NO: 1. In someembodiments, the region is within the complement of nucleotides72,892-72,972 of SEQ ID NO: 1. In some embodiments, the region is withinthe complement of nucleotides 72,902-72,972 of SEQ ID NO: 1.

In one embodiment, the oligomer can comprise a sequence selected fromthe group consisting of SEQ ID NOs: 804 to 900. In one embodiment, theoligomer (or contiguous nucleotide portion thereof) optionally has one,two, or three mismatches against the selected sequence. In anotherembodiment, the oligomer optionally comprises one or more nucleotideanalogs. In other embodiments, the oligomer has a design describedelsewhere herein (e.g., Section II.G.I, e.g., a gapmer design, e.g., analternating flank gapmer design) or a chemical structure describedelasewhere herein. Non-limiting examples of the nucleotide analogsuseful for the invention are disclosed elsewhere herein.

When the oligomer sequences are listed only with lower case letters(e.g., ctttatttccaaattcactt (SEQ ID NO: 676), the nucleic acids includedin the oligomer can be either naturally occurring nucleic acids ornucleotide analogs. If an oligomer sequence is described as acombination of lower case letters and upper case letters (e.g.,CTTtatttccaaattcaCTT, SEQ ID NO: 961), the upper case letters in thesequence are nucleotide analogs (e.g, LNA) while the lower case lettersare naturally occurring nucleic acids (e.g., DNA). Therefore, forexample, when a sequence “CTTtatttccaaattcaCTT” (SEQ ID NO: 961) isprovided herein, also provided is “ctttatttccaaattcactt (or SEQ ID NO:676), wherein the three nucleic acids at the 3′ end are nucleotideanalogs (e.g., LNA) and the three nucleic acids at the 5′ end arenaturally occurring nucleic acids (e.g., DNA)” or “ctttatttccaaattcactt(or SEQ ID NO: 676) with a design of LLLDDDDDDDDDDDDDDLLL, wherein L isa nucleotide analog and D is a DNA unit.”

In certain embodiments, the oligomer of the invention comprises anucleotide sequence selected from SEQ ID NO: 4 to 953, 956 to 958, and960. See FIGS. 2, 3, 6, 7, 16A, 16B, 20A, and 20B. In certainembodiments, the oligomer of the invention comprises a nucleotidesequence selected from the sequences listed in FIGS. 2, 3, 6, 7, 16A,16B, 20A, and 20B. Nonetheless, the design of the oligomers is notlimited to the design shown in FIGS. 2, 3, 6, 7, 16A, 16B, 20A, and 20B.The oligomers of the invention can have any oligomer design, e.g.,gapmer, mixmer, blockmer, or fully modified, as described elsewhereherein. Thus, in some embodiments, the oligomer of the inventioncomprises a nucleotide sequence selected from SEQ ID NO: 4 to 953, 956to 958, and 960, wherein at least one nucleotide is modified. In otherembodiments, the oligomer of the invention comprises a nucleotidesequence selected from SEQ ID NOs: 4 to 953, 956 to 958, and 960,wherein the one to five nucleotides at the 5′ end and the one to fivenucleotides at the 3′ end are nucleotide analogs (e.g., LNA) and theother nucleotides in the middle are naturally occurring nucleic acids.In still other embodiments, the oligomer comprises a nucleotide sequenceselected from SEQ ID NO: 4 to 953, 956 to 958, and 960, wherein thenucleotide design for the oligomer is as described in FIGS. 2, 3, 6, and7 (the upper case letter indicates a nucleotide analog, e.g., LNA, andthe lower case letter indicates a naturally occurring nucleic acid(e.g., DNA). In yet other embodiments, the oligomer of the inventioncomprises a nucleotide sequence selected from SEQ ID NOs: 4 to 953, 956to 958, and 960, wherein the backbone comprises at least onephosphorothioate bond. In a particular embodiment, the oligomercomprises a nucleotide sequence selected from the sequences in FIG. 7,e.g., SEQ ID NOs: 677, 679, 715, 681 644, 647, 593, 716, 474, 683, 587,685, 646, 680, 201, 473, 645, 532, 538, 535, 650, 533, 590, 7, 153, 686,471, 223, 688, 53, 154, 202, 595, 655, 482, 227, 485, 589, 370, 548,250, 251, 258, 256, 51, 69, 71, 255, 84, 262, 365, 285, 392, 417, 76,74, 390, 28, 46, 43, 49, 52, 67, 56, 60, 698, 773, 782, 684, 784, 780,786, 795, 798, 794, 797, 796, 705, 592, 472, 720, 745, 691, 687, 690,740, 724, 695, 689, 741, 714, 726, 799, 484, 801, 536, 800, 543, 545,537, 476, 528, 477, 479, 487, 467, 602, 594, 604, 603, 529, 530, 598,527, 539, 481, 480, 469, 540, 600, 486, 601, 531, 588, 586, 542, 596,544, 468, 653, 591, 534, 470, 547, 478, 546, 648, 541, 466, 599, 483,597, and 475, wherein the oligomer is designed as described in FIG. 7,and wherein the upper case letters are nucleotide analogs, e.g., LNAs,and the lower case letters are DNAs. Non-limiting examples of theoligomers are shown in FIGS. 2, 3, 6, 7, 16A, and 16B. In someembodiments, the oligomers of the invention bind to the target nucleicacid sequence (e.g., MAPT transcript) and inhibit or reduce expressionof the MAPT transcript by at least 10% or 20% compared to the normal(i.e., control) expression level in the cell, e.g., at least 30%, 40%,50%, 60%, 70%, 80%, 90% or 95% compared to the normal expression level(such as the expression level in the absence of the oligomer(s) orconjugate(s)) in the cell.

In certain embodiments, the oligomers of the invention bind to the MAPTtranscript and inhibit or reduce expression of the MAPT mRNA by at leastabout 10% or about 20% compared to the normal (i.e. control) expressionlevel in the cell, e.g., at least about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90% or about 95% compared to the normalexpression level (such as the expression level in the absence of theoligomer(s) or conjugate(s)) in the cell. In certain embodiments, theoligomer reduces expression of Tau protein in a cell followingadministration of the oligomer by at least 60%, at least 70%, at least80%, or at least 90% compared to a cell not exposed to the oligomer(i.e., control). In some embodiments, the oligomer reduces expression ofTau protein in a cell following administration of the oligomer by atleast about 60%, at least about 70%, at least about 80%, or at leastabout 90% compared to a cell not exposed to the oligomer (i.e.,control).

In certain embodiments, the oligomer of the invention has at least oneproperty selected from: (1) reduces expression of Tau mRNA in a cell,compared to a control cell that has not been exposed to the oligomer;(2) does not significantly reduce calcium oscillations in a cell; (3)does not significantly reduce tubulin intensity in a cell; (4) reducesexpression of Tau protein in a cell; and (5) any combinations thereofcompared to a control cell that has not been exposed to the oligomer.

In some embodiments, the oligomer of the invention does notsignificantly reduce calcium oscillations in a cell, e.g., neuronalcells. If the oligomer does not significantly reduce calciumoscillations in a cell, this property of the oligomer corresponds with areduced neurotoxicity of the oligomer. In some embodiments, calciumoscillations are greater than or equal to 95%, greater than or equal to90%, greater than or equal to 85%, greater than or equal to 80%, greaterthan or equal to 75%, or greater than or equal to 70% of oscillations ina cell not exposed to the oligomer.

Calcium oscillations are important for the proper functions of neuronalcells. Networks of cortical neurons have been shown to undergospontaneous calcium oscillations resulting in the release of theneurotransmitter glutamate. Calcium oscillations can also regulateinteractions of neurons with associated glia, in addition to otherassociated neurons in the network, to release other neurotransmitters inaddition to glutamate. Regulated calcium oscillations are required forhomeostasis of neuronal networks for normal brain function. (See,Shashank et al., Brain Research, 1006(1): 8-17 (2004); Rose et al.,Nature Neurosci., 4:773-774 (2001); Zonta et al., J Physiol Paris.,96(3-4):193-8 (2002); Pasti et al., J. Neurosci., 21(2): 477-484(2001).) Glutamate also activates two distinct ion channels,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorsand N-methyl-D-aspartate (NMDA) receptors.

In some embodiments, the calcium oscillations measured in the presentmethods are AMPA-dependent calcium oscillations. In some embodiments,the calcium oscillations are NMDA-dependent calcium oscillations. Insome embodiments, the calcium oscillations are gamma-aminobutyric acid(GABA)-dependent calcium oscillations. In some embodiments, the calciumoscillations can be a combination of two or more of AMPA-dependent,NMDA-dependent or GABA-dependent calcium oscillations.

In certain embodiments, the calcium oscillations measured in the presentmethods are AMPA-dependent calcium oscillations. In order to measureAMPA-dependent calcium oscillations, the calcium oscillations can bemeasured in the presence of Mg²⁺ ions (e.g., MgCl₂). In certainembodiments, the method further comprises adding Mg²⁺ ions (e.g., MgCl₂)at an amount that allows for detection of AMPA-dependent calciumoscillations. In some embodiments, the effective ion concentrationallowing for detection of AMPA-dependent calcium oscillations is atleast about 0.5 mM. In other embodiments, the effective ionconcentration to induce AMPA-dependent calcium oscillations is at leastabout 0.6 mM, at least about 0.7 mM, at least about 0.8 mM, at leastabout 0.9 mM, at least about 1 mM, at least about 1.5 mM, at least about2.0 mM, at least about 2.5 mM, at least about 3.0 mM, at least about 4mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, atleast about 8 mM, at least about 9 mM, or at least about 10 mM. In aparticular embodiment, the concentration of Mg²⁺ ions (e.g., MgCl₂)useful for the methods is 1 mM. In certain embodiments, theconcentration of Mg²⁺ ions (e.g., MgCl₂) useful for the present methodsis about 1 mM to about 10 mM, about 1 mM to about 15 mM, about 1 mM toabout 20 mM, or about 1 mM to about 25 mM. Mg²⁺ ions can be added by theaddition of magnesium salts, such as magnesium carbonate, magnesiumchloride, magnesium citrate, magnesium hydroxide, magnesium oxide,magnesium sulfate, and magnesium sulfate heptahydrate.

In some embodiments, calcium oscillations are measured in the presentmethod through the use of fluorescent probes which detect thefluctuations of intracellular calcium levels. For example, detection ofintracellular calcium flux can be achieved by staining the cells withfluorescent dyes which bind to calcium ions (known as fluorescentcalcium indicators) with a resultant, detectable change in fluorescence(e.g., Fluo-4 AM and Fura Red AM dyes available from Molecular Probes.Eugene, Oreg., United States of America).

In other embodiments, the oligomers of the invention do notsignificantly reduce the tubulin intensity in a cell. In someembodiments, tubulin intensity is greater than or equal to 95%, greaterthan or equal to 90%, greater than or equal to 85%, greater than orequal to 80%, greater than or equal to 75%, or greater than or equal to70% of tubulin intensity in a cell not exposed to the oligomer (orexposed to saline).

In some embodiments, such property is observed when using from 0.04 nMto 400 μM concentration of the oligomer of the invention. In the same ora different embodiment, the inhibition or reduction of expression ofMAPT mRNA and/or Tau protein in the cell results in less than 100%, suchas less than 98%, less than 95%, less than 90%, less than 80%, such asless than 70%, mRNA or protein levels compared to cells not exposed tothe oligomer. Modulation of expression level can be determined bymeasuring Tau protein levels, e.g., by methods such as SDS-PAGE followedby western blotting using suitable antibodies raised against the targetprotein. Alternatively, modulation of expression levels can bedetermined by measuring levels of MAPT mRNA, e.g., by northern blot orquantitative RT-PCR. When measuring inhibition via mRNA levels, thelevel of down-regulation when using an appropriate dosage, such as fromabout 0.04 nM to about 400 μM concentration, is, in some embodimentstypically to a level of from about 10-20% the normal levels in the cellin the absence of the oligomer.

In certain embodiments, the oligomer of the invention has an in vivotolerability less than or equal to a total score of 4, wherein the totalscore is the sum of a unit score of five categories, which are 1)hyperactivity; 2) decreased activity and arousal; 3) motor dysfunctionand/or ataxia; 4) abnormal posture and breathing; and 5) tremor and/orconvulsions, and wherein the unit score for each category is measured ona scale of 0-4. In certain embodiments, the in vivo tolerability is lessthan or equal to the total score of 3, the total score of 2, the totalscore of 1, or the total score of 0. In some embodiment, the assessmentfor in vivo tolerability is determined as described in Example 5 below.

In some embodiments, the oligomer can tolerate 1, 2, 3, or 4 (or more)mismatches, when hybridizing to the target sequence and stillsufficiently bind to the target to show the desired effect, i.e.,down-regulation of the target mRNA and/or protein. Mismatches can, forexample, be compensated by increased length of the oligomer nucleotidesequence and/or an increased number of nucleotide analogs, which aredisclosed elsewhere herein.

In some embodiments, the oligomer of the invention comprises no morethan 3 mismatches when hybridizing to the target sequence. In otherembodiments, the contiguous nucleotide sequence comprises no more than 2mismatches when hybridizing to the target sequence. In otherembodiments, the contiguous nucleotide sequence comprises no more than 1mismatch when hybridizing to the target sequence. In some embodiments,the target sequence is a region within nucleotides 134,947-138,940 ofSEQ ID NO: 1. In some embodiments, the contiguous nucleotide sequencecomprises no more than a single mismatch when hybridizing to the targetsequence, a region within nucleotides 134,947-138,940 of SEQ ID NO: 1.In some embodiments, the target sequence is a region within nucleotides135,050-138,940 of SEQ ID NO: 1. In some embodiments, the contiguousnucleotide sequence comprises no more than a single mismatch whenhybridizing to the target sequence, a region within nucleotides135,050-138,940 of SEQ ID NO: 1. In some embodiments, the targetsequence is a region within nucleotides 72,802-73,072 of SEQ ID NO: 1.In some embodiments, the contiguous nucleotide sequence comprises nomore than a single mismatch when hybridizing to the target sequence, aregion within nucleotides 72,802-73,072 of SEQ ID NO: 1.

In some embodiments the region within the complement or the region canconsist of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, or 29 contiguous nucleotides, such as from 12-22, such asfrom 14-21 nucleotides. Suitably, in some embodiments, the region is ofthe same length as the contiguous nucleotide sequence of the oligomer ofthe invention.

In some embodiments the oligomer according to the invention comprises anucleotide sequence, or a region within the sequence, according to anyone of SEQ ID NOs: 4 to 953, 956 to 958, and 960.

In other embodiments the oligomer according to the invention comprises anucleotide sequence, or a region within the sequence, according to anyone of SEQ ID NOs: 804 to 900. In other embodiments the oligomeraccording to the invention comprises a nucleotide sequence, or a regionwithin the sequence, according to any one of SEQ ID NOs: 936 to 953,956to 958, and 960.

However, it is recognized that, in some embodiments, the nucleotidesequence of the oligomer can comprise additional 5′ or 3′ nucleotides,such as, independently, 1, 2, 3, 4 or 5 additional nucleotides 5′ and/or3′, which are non-complementary to the target sequence. In this respectthe oligomer of the invention, can, in some embodiments, comprise acontiguous nucleotide sequence which is flanked 5′ and/or 3′ byadditional nucleotides. In some embodiments the additional 5′ and/or 3′nucleotides are naturally occurring nucleotides, such as DNA or RNA.

In some embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 677 (e.g., ASO-000757), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 679(e.g., ASO-001928), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 715 (e.g., ASO-001962), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 681 (e.g., ASO-001921), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 644(e.g., ASO-000756), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 647 (e.g., ASO-001948), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 593 (e.g., ASO-001941), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 716(e.g., ASO-001956), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 474 (e.g., ASO-001919), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 683 (e.g., ASO-001942), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 587(e.g., ASO-000755), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 685 (e.g., ASO-001935), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 472 (e.g., ASO-001940), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 646(e.g., ASO-001955), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 680 (e.g., ASO-001968) or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 201 (e.g., ASO-000662), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 473(e.g., ASO-001933), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 645 (e.g., ASO-001967), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 532 (e.g., ASO-001954), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 538(e.g., ASO-001960), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 535 (e.g., ASO-001966), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 650 (e.g., ASO-001961), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 533(e.g., ASO-001947), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 590 (e.g., ASO-001920), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 7 (e.g., ASO-000829), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 740(e.g., ASO-002007), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 714 (e.g., ASO-002012), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 487 (e.g., ASO-002038), or aregion thereof.

In some embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 153 (e.g., ASO-000540), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 686(e.g., ASO-000013), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 471 (e.g., ASO-000753), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 223 (e.g., ASO-000642), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 688(e.g., ASO-000762), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 53 (e.g., ASO-000389), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 154 (e.g., ASO-000555), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 202(e.g., ASO-000566), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 595 (e.g., ASO-001934), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 655 (e.g., ASO-000761), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 482(e.g., ASO-001926), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 485 (e.g., ASO-000758), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 589 (e.g., ASO-000760), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 370(e.g., ASO-000635), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 548 (e.g., ASO-000759), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 250 (e.g., ASO-000388), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 251(e.g., ASO-000390), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 258 (e.g., ASO-000394), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 256 (e.g., ASO-000396), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 51(e.g., ASO-000411), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 69 (e.g., ASO-000435), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 71 (e.g., ASO-000442), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 255(e.g., ASO-000447), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 84 (e.g., ASO-000449), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 262 (e.g., ASO-000451), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 365(e.g., ASO-000468), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 285 (e.g., ASO-000478), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 392 (e.g., ASO-000527), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 417(e.g., ASO-000543), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 76 (e.g., ASO-000558), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 74 (e.g., ASO-000581), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 390(e.g., ASO-000614), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 28 (e.g., ASO-000830), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 46 (e.g., ASO-001778), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 43(e.g., ASO-001779), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 49 (e.g., ASO-001780), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 52 (e.g., ASO-001781), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 67(e.g., ASO-001782), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 56 (e.g., ASO-001925, or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 60 (e.g., ASO-001953), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 698(e.g., ASO-214296), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 773 (e.g., ASO-000118), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 782 (e.g., ASO-000125), or aregion thereof.

In some embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 807 (e.g., ASO-000461), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 824(e.g., ASO-001783), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 825 (e.g., ASO-001784), or a region thereof. Insome embodiments the oligomer according to the invention comprises anucleotide sequence according to SEQ ID NO: 811 (e.g., ASO-000520), or aregion thereof. In some embodiments the oligomer according to theinvention comprises a nucleotide sequence according to SEQ ID NO: 818(e.g., ASO-000774), or a region thereof. In some embodiments theoligomer according to the invention comprises a nucleotide sequenceaccording to SEQ ID NO: 817 (e.g., ASO-000773), or a region thereof.

In some embodiments, the oligomer of the invention has a sequence scoregreater than or equal to 0.2, wherein the sequence score is calculatedby formula I:

$\begin{matrix}{\frac{{\#\mspace{14mu}{of}\mspace{14mu} C\mspace{14mu}{nucleotides}\mspace{14mu}{and}\mspace{14mu}{analogs}\mspace{14mu}{thereof}} - {\#\mspace{14mu}{of}\mspace{14mu} G\mspace{14mu}{nucleotides}\mspace{14mu}{and}\mspace{14mu}{analogs}\mspace{14mu}{thereof}}}{{Total}\mspace{14mu}{nucleotide}\mspace{14mu}{length}}.} & (I)\end{matrix}$

In other embodiments, the oligomer of the invention has a sequence scoregreater than or equal to 0.2, wherein the sequence score is calculatedby formula IA:

$\begin{matrix}{\frac{\begin{matrix}{{\#\mspace{14mu}{of}\mspace{14mu} C\mspace{14mu}{nucleotides}\mspace{14mu}{and}\mspace{14mu} 5\text{-}{methylcytosine}\mspace{14mu}{nucleotides}} -} \\{\#\mspace{14mu}{of}\mspace{14mu} G\mspace{14mu}{nucleotides}}\end{matrix}}{{Total}\mspace{14mu}{nucleotide}\mspace{14mu}{length}}.} & ({IA})\end{matrix}$

In these embodiments, a sequence score of greater than or equal to a cutoff value corresponds to a reduced neurotoxicity of the oligomer.

In certain embodiments, the oligomer of the invention has a sequencescore greater than or equal to about 0.1, 0.2, 0.25, 0.3, 0.35, 0.4,0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0. Inone embodiment, the oligomer of the invention comprises a contiguousnucleotide sequence hybridizing to a non-coding region of a MAPTtranscript, wherein the sequence score of the oligomer is greater thanor equal to about 0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6,0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0. In another embodiment,the oligomer of the invention comprises a contiguous nucleotide sequencehybridizing to a 3′ UTR of a MAPT transcript, wherein the sequence scoreof the oligomer is greater than or equal to about 0.1, 0.2, 0.25, 0.3,0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95,or 1.0. In another embodiment, the oligomer of the invention comprises acontiguous nucleotide sequence hybridizing to a 5′ UTR of a MAPTtranscript, wherein the sequence score of the oligomer is greater thanor equal to about 0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6,0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0. In another embodiment,the oligomer of the invention comprises a contiguous nucleotide sequencehybridizing to exon 2 of a MAPT transcript, wherein the sequence scoreof the oligomer is greater than or equal to about 0.1, 0.2, 0.25, 0.3,0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95,or 1.0. In all of these embodiments, when the sequence score is greaterthan or equal to the cut off value, the oligomer is considered to havereduced neurotoxicity.

II.C. Oligomer Length

The oligomers can comprise a contiguous nucleotide sequence of a totalof 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or 50 contiguous nucleotides in length.

In some embodiments, the oligomers comprise a contiguous nucleotidesequence of a total of about 10-22, such as 10-21 or 12-18, such as13-17 or 12-16, such as 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguousnucleotides in length.

In some embodiments, the oligomers comprise a contiguous nucleotidesequence of a total of 10, 11, 12, 13, or 14 contiguous nucleotides inlength.

In some embodiments, the oligomer according to the invention consists ofno more than 22 nucleotides, such as no more than 21 or 20 nucleotides,such as no more than 18 nucleotides, such as 15, 16 or 17 nucleotides.In some embodiments the oligomer of the invention comprises less than 22nucleotides. It should be understood that when a range is given for anoligomer, or contiguous nucleotide sequence length, the range includesthe lower and upper lengths provided in the range, for example from (orbetween) 10-50, includes both 10 and 50.

II.D. Nucleosides and Nucleoside Analogs

In one aspect of the invention, the oligomers comprise one or morenon-naturally occurring nucleotide analogs. “Nucleotide analogs” as usedherein are variants of natural nucleotides, such as DNA or RNAnucleotides, by virtue of modifications in the sugar and/or basemoieties. Analogs could in principle be merely “silent” or “equivalent”to the natural nucleotides in the context of the oligonucleotide, i.e.have no functional effect on the way the oligonucleotide works toinhibit target gene expression. Such “equivalent” analogs cannevertheless be useful if, for example, they are easier or cheaper tomanufacture, or are more stable to storage or manufacturing conditions,or represent a tag or label. In some embodiments, however, the analogswill have a functional effect on the way in which the oligomer works toinhibit expression; for example by producing increased binding affinityto the target and/or increased resistance to intracellular nucleasesand/or increased ease of transport into the cell. Specific examples ofnucleoside analogs are described by e.g. Freier & Altmann; Nucl. AcidRes., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in DrugDevelopment, 2000, 3(2), 293-213, and in Scheme 1:

In one embodiment, the oligomer includes at least one, at least two, atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, or at least ten nucleotide analogs. Inanother embodiment, the oligomer includes four, six, eight, or tennucleotide analogs.

Examples of the nucleotide analogs include, but are not limited to,Locked Nucleic Acid (LNA); 2′-O-alkyl-RNA; 2′-amino-DNA; 2′-fluoro-DNA;arabino nucleic acid (ANA); 2′-fluoro-ANA, hexitol nucleic acid (HNA),intercalating nucleic acid (INA), constrained ethyl nucleoside (cEt),2′-O-methyl nucleic acid (2′-OMe), 2′-O-methoxyethyl nucleic acid(2′-MOE), or any combination thereof.

“Hexitol nucleic acids” or “HNA” are composed of phosphorylated2,3-dideoxy-D-arabino-hexitol units with a nucleobase situated in the2-[S]-position.

“cEt” or “constrained ethyl” means a bicyclic nucleoside having a sugarmoiety comprising a bridge connecting the 4′-carbon and the 2′-carbon,wherein the bridge has the formula: 4′-CH(CH3)-0-2′.

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH2)2-OCH3 and MOE) refers toan O-methoxy-ethyl modification at the 2′ position of a furanosyl ring.A 2′-O-methoxyethyl modified sugar is a modified sugar.

As used herein, “2′-F” refers to modification of the 2′ position of thefuranosyl sugar ring to comprise a fluoro group.

As used herein, “2′-OMe” or “2′-OCH3” or “2′-O-methyl” each refers tomodification at the 2′ position of the furanosyl sugar ring to comprisea —OCH3 group.

The oligomer can thus comprise a simple sequence of natural occurringnucleotides—for example, 2′-deoxynucleotides (referred to hereingenerally as “DNA”), but also possibly ribonucleotides (referred toherein generally as “RNA”), or a combination of such naturally occurringnucleotides and one or more non-naturally occurring nucleotides, i.e.nucleotide analogs. Such nucleotide analogs can suitably enhance theaffinity of the oligomer for the target sequence.

Examples of suitable nucleotide analogs are provided by WO2007/031091,which is incorporated by reference in its entirety, or are referencedtherein.

Incorporation of affinity-enhancing nucleotide analogs in the oligomer,such as LNA or 2′-substituted sugars, can allow the size of thespecifically binding oligomer to be reduced, and can also reduce theupper limit to the size of the oligomer before non-specific or aberrantbinding takes place.

In some embodiments, the oligomer comprises at least one LNA. Additionaldetails of the LNA compound are disclosed elsewhere herein. In someembodiments the oligomer comprises at least 2 LNAs. In some embodiments,the oligomer comprises from 3-10 LNAs, e.g., 6 or 7 LNAs, e.g., at least3 or at least 4, or at least 5, or at least 6, or at least 7, or 8 LNAs.In some embodiments all the nucleotides analogs can be LNA.

In a specific embodiment, the oligomer of the invention includes abicyclic sugar. Non-limiting examples of the bicyclic sugar includescEt, 2′,4′-constrained 2′-O-methoxyethyl (cMOE), LNA, α-LNA, β-LNA,2′-O,4′-C-ethylene-bridged nucleic acids (ENA), amino-LNA, oxy-LNA, orthio-LNA.

The term “thio-LNA” comprises a locked nucleotide in which Y in generalFormula III below is selected from S or —CH₂—S—. Thio-LNA can be in bothbeta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which Y in generalFormula III below is selected from —N(H)—, N(R)—, CH₂—N(H)—, and—CH₂—N(R)— where R is selected from hydrogen and C₁₋₄-alkyl. Amino-LNAcan be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which Y in generalFormula III below represents —O—. Oxy-LNA can be in both beta-D andalpha-L-configuration.

The term “ENA” comprises a locked nucleotide in which Y in generalFormula III below is —CH₂—O— (where the oxygen atom of —CH₂—O— isattached to the 2′-position relative to the base B). R^(e) is hydrogenor methyl.

In some exemplary embodiments LNA is selected from beta-D-oxy-LNA,alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particularbeta-D-oxy-LNA.

It will be recognized that when referring to a nucleotide sequence motifor nucleotide sequence, which consists of only nucleotides, theoligomers of the invention which are defined by that sequence, cancomprise a corresponding nucleotide analog in place of one or more ofthe nucleotides present in the sequence, such as LNA units or othernucleotide analogs, including cEt, cMOE, α-LNA, β-LNA, ENA, amino-LNA,oxy-LNA, thio-LNA, which raise the duplex stability/T_(m) of theoligomer/target duplex (i.e. affinity enhancing nucleotide analogs).

In some embodiments, any mismatches between the nucleotide sequence ofthe oligomer and the target sequence are found in regions outside theaffinity enhancing nucleotide analogs, such as region B as referred toherein, and/or region D as referred to herein, and/or at the site ofnon-modified such as DNA nucleotides in the oligonucleotide, and/or inregions which are 5′ or 3′ to the contiguous nucleotide sequence.

Examples of such modification of the nucleotide include modifying thesugar moiety to provide a 2′-substituent group or to produce a bridged(locked nucleic acid) structure which enhances binding affinity and canalso provide increased nuclease resistance.

In one embodiment, a nucleotide analog is oxy-LNA (such asbeta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such asbeta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (such asbeta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA andalpha-L-ENA). In a particular embodiment, a nucleotide analog isbeta-D-oxy-LNA.

In some embodiments the nucleotide analogs present within the oligomerof the invention (such as in regions A and C mentioned herein) areindependently selected from, for example: 2′-O-alkyl-RNA units,2′-amino-DNA units, 2′-fluoro-DNA units, LNA units, arabino nucleic acid(ANA) units, 2′-fluoro-ANA units, HNA units, INA (intercalating nucleicacid—Christensen, 2002. Nucl. Acids. Res. 2002 30: 4918-4925, herebyincorporated by reference) units and 2′-MOE units. In some embodimentsthere is only one of the above types of nucleotide analogs present inthe oligomer of the invention, or contiguous nucleotide sequencethereof.

In some embodiments the nucleotide analogs are 2′-O-methoxyethyl-RNA(2′-MOE), 2′-fluoro-DNA monomers, LNA nucleotide analogs, cEt, cMOE,α-LNA, β-LNA, ENA, amino-LNA, oxy-LNA, or thio-LNA units, and as suchthe oligonucleotide of the invention can comprise nucleotide analogswhich are independently selected from these types of analog, or cancomprise only one type of analog selected from those above. In someembodiments at least one of the nucleotide analogs is 2′-MOE-RNA, suchas 2, 3, 4, 5, 6, 7, 8, 9 or 10 2′-MOE-RNA nucleotide units. In someembodiments at least one of the nucleotide analogs is 2′-fluoro DNA,such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2′-fluoro-DNA nucleotide units.

In some embodiments, the oligomer according to the invention comprisesat least one Locked Nucleic Acid (LNA) unit, such as 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 LNA units, such as from 3-7 or 4-8 LNA units, or 3, 4, 5,6, 7, or 8 LNA units. In some embodiments, all the nucleotide analogsare LNA. In some embodiments, the oligomer can comprise bothbeta-D-oxy-LNA, and one or more of the following LNA units: thio-LNA,amino-LNA, oxy-LNA, and/or ENA in either the beta-D or alpha-Lconfigurations or combinations thereof. In some embodiments all LNAcytosine units are 5′-methylcytosine. In some embodiments of theinvention, the oligomer can comprise both LNA and DNA units. In certainembodiments, the combined total of LNA and DNA units is 10-50, 10-30,such as 10-25, e.g., 10-22, such as 10-21. In some embodiments of theinvention, the nucleotide sequence of the oligomer, such as thecontiguous nucleotide sequence consists of at least one LNA and theremaining nucleotide units are DNA units. In some embodiments theoligomer comprises only LNA nucleotide analogs and naturally occurringnucleotides (such as RNA or DNA, e.g., DNA nucleotides), optionally withmodified internucleotide linkages such as phosphorothioate.

The term “nucleobase” refers to the base moiety of a nucleotide andcovers both naturally occurring as well as non-naturally occurringvariants. Thus, “nucleobase” covers not only the known purine andpyrimidine heterocycles but also heterocyclic analogs and tautomeresthereof.

Examples of nucleobases include, but are not limited to adenine,guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine,5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine.

In some embodiments, at least one of the nucleobases present in theoligomer is a modified nucleobase selected from the group consisting of5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine.

In certain embodiments, the present invention includes oligomerscomprising nucleotide analogs. In some embodiments, the nucleotideanalog comprises a modified nucleobase such as 5-methylcytosine. Inother embodiments, the nucleotide analog comprise a modified nucleobasessuch as 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine. In certain embodiments, the oligomers have achemical structure as disclosed in FIG. 2 or FIG. 16B.

II.E LNA

The term “LNA” refers to a bicyclic nucleoside analog, known as “LockedNucleic Acid”. It can refer to an LNA monomer, or, when used in thecontext of an “LNA oligonucleotide,” LNA refers to an oligonucleotidecontaining one or more such bicyclic nucleotide analogs. LNA nucleotidesare characterized by the presence of a linker group (such as a bridge)between C2′ and C4′ of the ribose sugar ring—for example as shown as thebiradical R^(4*)-R^(2*) as described below.

In certain embodiments, the LNA used in the oligonucleotide compounds ofthe invention has the structure of the general formula V:

wherein for all chiral centers, asymmetric groups can be found in eitherR or S orientation;wherein X is selected from —O—, —S—, —N(RN*)—, —C(R6R6*)-, such as, insome embodiments —O—;

B is selected from hydrogen, optionally substituted C1-4-alkoxy,optionally substituted C1-4-alkyl, optionally substituted C1-4-acyloxy,nucleobases including naturally occurring and nucleobase analogs, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands; in someembodiments, B is a nucleobase or nucleobase analog;

P designates an internucleotide linkage to an adjacent monomer, or a5′-terminal group, such internucleotide linkage or 5′-terminal groupoptionally including the sub stituent R5 or equally applicable thesubstituent R5*;

P* designates an internucleotide linkage to an adjacent monomer, or a3′-terminal group;

R4* and R2* together designate a bivalent linker group consisting of 1-4groups/atoms selected from —C(RaRb)—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —O—,—Si(Ra)2-, —S—, —SO2-, —N(Ra)—, and >C═Z, wherein Z is selected from—O—, —S—, and —N(Ra)—, and Ra and Rb each is independently selected fromhydrogen, optionally substituted C1-12-alkyl, optionally substitutedC2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, optionallysubstituted C1-12-alkoxy, C2-12-alkoxyalkyl, C2-12-alkenyloxy, carboxy,C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C1-6-alkyl)amino, carbamoyl, mono- anddi(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- anddi(C1-6-alkyl)amino-C 1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino,carbamido, C1-6-alkanoyloxy, sulphono, C1-6-al kylsulphonyloxy, nitro,azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl can beoptionally substituted and where two geminal substituents Ra and Rbtogether can designate optionally substituted methylene (═CH2), whereinfor all chiral centers, asymmetric groups can be found in either R or Sorientation, and;

each of the substituents R1*, R2, R3, R5, R5*, R6 and R6*, which arepresent is independently selected from hydrogen, optionally substitutedC1-12-alkyl, optionally substituted C2-12-alkenyl, optionallysubstituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkoxyalkyl,C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,hetero-aryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C1-6-alkyl)amino, carbamoyl, mono- anddi(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- anddi(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino,carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro,azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl can beoptionally substituted, and where two geminal substituents together candesignate oxo, thioxo, imino, or optionally substituted methylene;wherein RN is selected from hydrogen and C1-4-alkyl, and where twoadjacent (non-geminal) substituents can designate an additional bondresulting in a double bond; and RN*, when present and not involved in abiradical, is selected from hydrogen and C1-4-alkyl; and basic salts andacid addition salts thereof. For all chiral centers, asymmetric groupscan be found in either R or S orientation.

In some embodiments, R4* and R2* together designate a biradicalconsisting of a groups selected from the group consisting ofC(RaRb)—C(RaRb)—, C(RaRb)—O—, C(RaRb)—NRa—, C(RaRb)—S—, andC(RaRb)—C(RaRb)—O—, wherein each Ra and Rb can optionally beindependently selected. In some embodiments, Ra and Rb can be,optionally independently selected from the group consisting of hydrogenand C1-6alkyl, such as methyl, such as hydrogen.

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—CH(CH₂OCH₃)— (2′O-methoxyethyl bicyclic nucleic acid—Seth at al.,2010, J. Org. Chem)—in either the R— or S— configuration. In someembodiments, R^(4*) and R^(2*) together designate the biradical—O—CH(CH₂CH₃)— (2′-ethyl bicyclic nucleic acid—Seth at al., 2010, J.Org. Chem).—in either the R— or S— configuration.

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—CH(CH₃)—.—in either the R— or S— configuration. In some embodiments,R^(4*) and R^(2*) together designate the biradical —O—CH₂—O—O—CH₂—(Sethat al., 2010, J. Org. Chem).

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—NR—CH₃—(Seth at al., 2010, J. Org. Chem).

In some embodiments, the LNA units have a structure selected from thefollowing group:

in which the orientation of the CH₃— substituent in the cEt LNA unitscan independently be R or S, and in which the orientation of the MeOCH₂—substituent in the cMOE LNA units can independently be R or S, and inwhich the orientation of the CH₃— substituent in the 5′-Me-LNA units canindependently be R or S.

In some embodiments, R^(1*), R², R³, R⁵, R^(5*) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆aminoalkyl. For all chiral centers, asymmetric groups can be found ineither R or S orientation.

In some embodiments, R^(1*), R², R³, R⁵, R^(5*) are hydrogen.

In some embodiments, R^(1*), R², R³ are independently selected from thegroup consisting of hydrogen, halogen, C₁₋₆ alkyl, substituted C₁₋₆alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl orsubstituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆ alkoxyl, acyl,substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆ aminoalkyl. Forall chiral centers, asymmetric groups can be found in either R or Sorientation.

In some embodiments, R^(1*), R², R³ are hydrogen.

In some embodiments, R⁵ and R^(5*) are each independently selected fromthe group consisting of H, —CH₃, —CH₂—CH₃, —CH₂—O—CH₃, and —CH=CH₂.Suitably in some embodiments, either R⁵ or R^(5*) are hydrogen, whereasthe other group (R⁵ or R^(5*) respectively) is selected from the groupconsisting of C₁₋₅ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, substituted C₁₋₆alkyl, substituted C₂₋₆ alkenyl, substituted C₂₋₆ alkynyl or substitutedacyl (—C(═O)—); wherein each substituted group is mono or polysubstituted with substituent groups independently selected from halogen,C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆alkenyl, C₂₋₆ alkynyl, substituted C₂₋₆ alkynyl, OJ₁, SJ₁, NJ₁J₂, N₃,COOJ₁, CN, O—C(═O)NJ₁J₂, N(H)C(═NH)NJ, J₂ or N(H)C(═X)N(H)J₂ wherein Xis O or S; and each J₁ and J₂ is, independently, H, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl, substituted C₂₋₆ alkynyl, C₁₋₆ aminoalkyl, substituted C₁₋₆aminoalkyl or a protecting group. In some embodiments either R⁵ orR^(5*) is substituted C₁₋₆ alkyl. In some embodiments either R⁵ orR^(5*) is substituted methylene wherein preferred substituent groupsinclude one or more groups independently selected from F, NJ₁J₂, N₃, CN,OJ₁, SJ₁, O—C(═O)NJ₁J₂, N(H)C(═NH)NJ, J₂ or N(H)C(O)N(H)J₂. In someembodiments each J₁ and J₂ is, independently H or C₁₋₆ alkyl. In someembodiments either R⁵ or R^(5*) is methyl, ethyl or methoxymethyl. Insome embodiments either R⁵ or R^(5*) is methyl. In a further embodimenteither R⁵ or R^(5*) is ethylenyl. In some embodiments either R⁵ orR^(5*) is substituted acyl. In some embodiments either R⁵ or R^(5*) isC(═O)NJ₁J₂. For all chiral centers, asymmetric groups can be found ineither R or S orientation. Such 5′ modified bicyclic nucleotides aredisclosed in WO 2007/134181, which is hereby incorporated by referencein its entirety.

In some embodiments B is a nucleobase, including nucleobase analogs andnaturally occurring nucleobases, such as a purine or pyrimidine, or asubstituted purine or substituted pyrimidine, such as a nucleobasereferred to herein, such as a nucleobase selected from the groupconsisting of adenine, cytosine, thymine, adenine, uracil, and/or amodified or substituted nucleobase, such as 5-thiazolo-uracil,2-thio-uracil, 5-propynyl-uracil, 2′thio-thymine, 5-methyl cytosine,5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6-diaminopurine.

In some embodiments, R^(4*) and R^(2*) together designate a biradicalselected from —C(R^(a)R^(b))—O—, —C(R^(a)R^(b))—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—O—,—C(R^(a)R^(b))—O—C(R^(c)R^(d))—, —C(R^(a)R^(b))—O—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—,—C(R^(a))═C(R^(b))—C(R^(c)R^(d))—, —C(R^(a)R^(b))—N(R^(c))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—N(R^(c))—, —C(R^(a)R^(b))—N(R^(c))—O—, and—C(R^(a)R^(b))—S—, —C(R^(a)R^(b))—C(R^(c)R^(d))—S—, wherein R^(a),R^(b), R^(c), R^(d), R^(e), and R^(f) each is independently selectedfrom hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl,hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy, carboxy,C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl can beoptionally substituted and where two geminal substituents R^(a) andR^(b) together can designate optionally substituted methylene (═CH₂).For all chiral centers, asymmetric groups can be found in either R or Sorientation.

In a further embodiment R^(4*) and R^(2*) together designate a biradical(bivalent group) selected from —CH₂—O—, —CH₂—S—, —CH₂—NH—, —CH₂—N(CH₃)—,—CH₂—CH₂—O—, —CH₂—CH(CH₃)—, —CH₂—CH₂—S—, —CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—O—, —CH₂—CH₂—CH(CH₃)—, —CH═CH—CH₂—, —CH₂—O—CH₂—O—,—CH₂—NH—O—, —CH₂—N(CH₃)—O—, —CH₂—O—CH₂—, —CH(CH₃)—O—, and—CH(CH₂—O—CH₃)—O—, and/or, —CH₂—CH₂—, and —CH═CH— For all chiralcenters, asymmetric groups can be found in either R or S orientation.

In some embodiments, R^(4*) and R^(2*) together designate the biradicalC(R^(a)R^(b))—N(R^(c))—O—, wherein R^(a) and R^(b) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆aminoalkyl, such as hydrogen, and; wherein R^(c) is selected from thegroup consisting of hydrogen, halogen, C₁₋₆ alkyl, substituted C₁₋₆alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl orsubstituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆ alkoxyl, acyl,substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆ aminoalkyl, suchas hydrogen.

In some embodiments, R^(4*) and R^(2*) together designate the biradicalC(R^(a)R^(b))—O—C(R^(c)R^(d))—O—, wherein R^(a), R^(b), R^(c), and R^(d)are independently selected from the group consisting of hydrogen,halogen, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substitutedC₂₋₆ alkenyl, C₂₋₆ alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl,substituted C₁₋₆ alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl orsubstituted C₁₋₆ aminoalkyl, such as hydrogen.

In some embodiments, R^(4*) and R^(2*) form the biradical —CH(Z)—O—,wherein Z is selected from the group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, substituted C₁₋₆ alkyl, substituted C₂₋₆ alkenyl,substituted C₂₋₆ alkynyl, acyl, substituted acyl, substituted amide,thiol or substituted thio; and wherein each of the substituted groups,is, independently, mono or poly substituted with optionally protectedsubstituent groups independently selected from halogen, oxo, hydroxyl,OJ₁, NJ₁J₂, SJ₁, N₃, OC(=X)J₁, OC(═X)NJ₁J₂, NJ³C(═X)NJ₁J₂ and CN,wherein each J₁, J₂ and J₃ is, independently, H or C₁₋₆ alkyl, and X isO, S or NJ₁. In some embodiments Z is C₁₋₆ alkyl or substituted C₁₋₆alkyl. In some embodiments Z is methyl. In some embodiments Z issubstituted C₁₋₆ alkyl. In some embodiments the substituent group isC₁₋₆ alkoxy. In some embodiments Z is CH₃OCH₂—. For all chiral centers,asymmetric groups can be found in either R or S orientation. Suchbicyclic nucleotides are disclosed in U.S. Pat. No. 7,399,845 which ishereby incorporated by reference in its entirety. In some embodiments,R^(1*), R², R³, R⁵, R^(5*) are hydrogen. In some embodiments, R^(1*),R², R^(3*) are hydrogen, and one or both of R⁵, R^(5*) can be other thanhydrogen as referred to above and in WO 2007/134181, which isincorporated by reference herein in its entirety.

In some embodiments, R^(4*) and R^(2*) together designate a biradicalwhich comprise a substituted amino group in the bridge such as consistof or comprise the biradical —CH₂—N(R^(c))—, wherein R^(c) is C₁₋₁₂alkyloxy. In some embodiments R^(4*) and R^(2*) together designate abiradical —Cq₃q₄-NOR—, wherein q₃ and q₄ are independently selected fromthe group consisting of hydrogen, halogen, C₁₋₆ alkyl, substituted C₁₋₆alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl orsubstituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆ alkoxyl, acyl,substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆ aminoalkyl;wherein each substituted group is, independently, mono or polysubstituted with substituent groups independently selected from halogen,OJ₁, SJ₁, NJ₁J₂, COOJ₁, CN, O—C(═O)NJ₁J₂, N(H)C(═NH)N J₁J₂ orN(H)C(═X═N(H)J₂ wherein X is O or S; and each of J₁ and J₂ is,independently, H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆aminoalkyl or a protecting group. For all chiral centers, asymmetricgroups can be found in either R or S orientation. Such bicyclicnucleotides are disclosed in WO2008/150729 which is hereby incorporatedby reference in its entirety. In some embodiments, R^(1*), R², R³, R⁵,R^(5*) are independently selected from the group consisting of hydrogen,halogen, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substitutedC₂₋₆ alkenyl, C₂₋₆ alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl,substituted C₁₋₆ alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl orsubstituted C₁₋₆ aminoalkyl. In some embodiments, R^(1*), R², R³, R⁵,R^(5*) are hydrogen. In some embodiments, R^(1*), R², R³ are hydrogenand one or both of R⁵, R^(5*) can be other than hydrogen as referred toabove and in WO 2007/134181. In some embodiments R^(4*) and R^(2*)together designate a biradical (bivalent group) C(R^(a)R^(b))—O—,wherein R^(a) and R^(b) are each independently halogen, C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl,C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substitutedC₁-C₁₂ alkoxy, OJ₁ SJ₁, SOJ₁, SO₂J₁, NJ₁J₂, N₃, CN, C(═O)OJ₁,C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═NH)NJ₁J₂, N(H)C(═O)NJ₁J₂ orN(H)C(═S)NJ₁J₂; or R^(a) and R^(b) together are ═C(q3)(q4); q₃ and q₄are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂alkyl; each substituted group is, independently, mono or polysubstituted with substituent groups independently selected from halogen,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, OJ₁, SJ₁, NJ₁J₂, N₃,CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ orN(H)C(═S)NJ₁J₂ and; each J₁ and J₂ is, independently, H, C1-C₆ alkyl,substituted C1-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, substituted C₂-C₆ alkynyl, C1-C₆ aminoalkyl, substituted C1-C₆aminoalkyl or a protecting group. Such compounds are disclosed inWO2009006478A, hereby incorporated in its entirety by reference.

In some embodiments, R^(4*) and R^(2*) form the biradical -Q-, wherein Qis C(q₁)(q₂)C(q₃)(q₄), C(q₁)=C(q₃), C[═C(q₁)(q₂)]-C(q₃)(q₄) orC(q₁)(q₂)-C[═C(q₃)(q₄)], q₁, q₂, q₃, q₄ are each independently. H,halogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,substituted C₁₋₁₂ alkoxy, OJ₁, SJ₁, SO₂J₁, SO₂J₁, NJ₁J₂, N₃, CN,C(═O)OJ₁, C(═O)—NJ₁J₂, C(⊚O) J₁, —C(═O)NJ₁J₂, N(H)C(═NH)NJ₁J₂,N(H)C(═O)NJ₁J₂ or N(H)C(═S)NJ₁J₂; each J₁ and J₂ is, independently, H,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ aminoalkyl or a protectinggroup; and, optionally wherein when Q is C(q₁)(q₂)(q₃)(q₄) and one of q₃or q₄ is CH₃ then at least one of the other of q₃ or q₄ or one of q₁ andq₂ is other than H. In some embodiments, R^(1*), R², R³, R⁵, R^(5*) arehydrogen. For all chiral centers, asymmetric groups can be found ineither R or S orientation. Such bicyclic nucleotides are disclosed inWO2008/154401 which is hereby incorporated by reference in its entirety.In some embodiments, R^(1*), R², R³, R⁵, R^(5*) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆aminoalkyl. In some embodiments, R^(1*), R², R³, R⁵, R^(5*) arehydrogen. In some embodiments, R^(1*), R², R³ are hydrogen and one orboth of R⁵, R^(5*) can be other than hydrogen as referred to above andin WO 2007/134181 or WO2009/067647 (alpha-L-bicyclic nucleic acidsanalogs).

Further bicyclic nucleoside analogs and their use in antisenseoligonucleotides are disclosed in WO2011/115818, WO2011/085102,WO2011/017521, WO09/100320, WO10/036698, WO09/124295 & WO09/006478, eachof which are incorporated by reference herein in their entireties. Suchnucleoside analogs can in some aspects be useful in the compounds ofpresent invention.

In some embodiments the LNA used in the oligonucleotide compounds of theinvention has the structure of the general formula VI:

wherein Y is selected from the group consisting of —O—, —CH₂O—, —S—,—NH—, N(R^(e)) and/or —CH₂—; Z and Z* are independently selected amongan internucleotide linkage, R^(H), a terminal group or a protectinggroup; B constitutes a natural or non-natural nucleotide base moiety(nucleobase), and R^(H) is selected from hydrogen and C₁₋₄-alkyl; R^(a),R^(b) R^(c), R^(d) and R^(e) are, optionally independently, selectedfrom the group consisting of hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl can beoptionally substituted and where two geminal substituents R^(a) andR^(b) together can designate optionally substituted methylene (═CH₂);and R^(H) is selected from hydrogen and C₁₋₄-alkyl. In some embodimentsR^(a), R^(b) R^(e), R^(d) and R^(e) are, optionally independently,selected from the group consisting of hydrogen and C₁₋₆ alkyl, such asmethyl. For all chiral centers, asymmetric groups can be found in eitherR or S orientation, for example, two exemplary stereochemical isomersinclude the beta-D and alpha-L isoforms, which can be illustrated asfollows:

Specific exemplary LNA units are shown below:

In other embodiments, the oligomers of the invention comprisenucleotides with modified sugar moieties as described in FIGS. 2, 3, 6,7, 16A, 16B, 20A or 20B.

II.F. RNase Recruitment

It is recognized that an oligomeric compound can function via non RNasemediated degradation of target mRNA, such as by steric hindrance oftranslation, or other methods, however, in one aspect, the oligomers ofthe invention are capable of recruiting an endoribonuclease (RNase),such as RNaseH.

In one aspect, the oligomer, or contiguous nucleotide sequence,comprises a region of at least 7 consecutive nucleotide units, such asat least 8 or at least 9 consecutive nucleotide units (residues), incertain embodiments including 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, or 23 consecutive nucleotides, which, when formed ina duplex with the complementary target RNA is capable of recruitingRNase. The contiguous sequence which is capable of recruiting RNase canbe region B as referred to in the context of a gapmer as describedherein. In some embodiments the size of the contiguous sequence which iscapable of recruiting RNase, such as region B, can be higher, such as10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotideunits.

U.S. Pat. No. 6,617,442, which is incorporated by reference herein inits entirety, provides in vitro methods for determining RNaseH activity,which can be used to determine the ability to recruit RNaseH. Therefore,in one embodiment, an oligomer of the invention is capable of recruitingRNaseH. In another embodiment, the invention includes a method ofidentifying an oligomer which is capable of utilizing RNaseH mechanism,e.g., recruiting RNaseH.

Oligomers can be screened to identify those which are effective inrecruiting RNaseH. The ability of oligomers to recruit RNaseH can bedetermined by measuring the binding of the oligomers to RNaseH. Themethods of determining binding of the oligomers to RNaseH are well knownin the art. For example, the oligomers can be radiolabeled and bindingof the oligomers to RNaseH can be detected by autoradiography. In someembodiments, fusion proteins of RNaseH with glutathione-S-transferase orsmall peptide tags can be prepared and immobilized to a solid phase suchas beads. Labeled or unlabeled oligomers to be screened for binding tothis enzyme can then be incubated with the solid phase. Oligomers whichbind to the enzyme immobilized to the solid phase can then be identifiedeither by detection of bound label or by eluting specifically the boundoligomers from the solid phase. Another method involves screening ofoligomer libraries for binding partners. Recombinant tagged or labeledRNaseH is used to select oligomers from the library which interact withthe enzyme. Sequencing of the oligomers leads to identification of thoseoligomers which will be more effective as antisense molecules.

An oligomer is deemed capable of recruiting RNaseH if, when providedwith the complementary RNA target, it has an initial rate, as measuredin pmol/l/min, of at least 1%, such as at least 5%, such as at least 10%or ,more than 20% of the of the initial rate determined using DNA onlyoligonucleotide, having the same base sequence but containing only DNAmonomers, with no 2′ substitutions, with phosphorothioate linkage groupsbetween all monomers in the oligonucleotide, using the methodologyprovided by Example 91-95 of U.S. Pat. No. 6,617,442.

In some embodiments, an oligomer is deemed essentially incapable ofrecruiting RNaseH if, when provided with the complementary RNA target,and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is lessthan 1%, such as less than 5%,such as less than 10% or less than 20% ofthe initial rate determined using the equivalent DNA onlyoligonucleotide, with no 2′ substitutions, with phosphorothioate linkagegroups between all nucleotides in the oligonucleotide, using themethodology provided by Example 91-95 of U.S. Pat. No. 6,617,442.

In other embodiments, an oligomer is deemed capable of recruiting RNaseHif, when provided with the complementary RNA target, and RNaseH, theRNaseH initial rate, as measured in pmol/l/min, is at least 20%, such asat least 40%, such as at least 60%, such as at least 80% of the initialrate determined using the equivalent DNA only oligonucleotide, with no2′ substitutions, with phosphorothioate linkage groups between allnucleotides in the oligonucleotide, using the methodology provided byExample 91-95 of U.S. Pat. No. 6,617,442.

Typically the region of the oligomer which forms the consecutivenucleotide units which, when formed in a duplex with the complementarytarget RNA is capable of recruiting RNase consists of nucleotide unitswhich form a DNA/RNA like duplex with the RNA target—and include bothDNA units and LNA units which are in the alpha-L configuration,particularly preferred being alpha-L-oxy LNA.

In some embodiments, the monomers which are capable of recruiting RNaseare selected from the group consisting of DNA monomers, alpha-L-LNAmonomers, C4′ alkylayted DNA monomers (see PCT/EP2009/050349 and Vesteret al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, herebyincorporated by reference in its entirety), and UNA (unlinked nucleicacid) nucleotides (see Fluiter et al., Mol. Biosyst., 2009, 10, 1039,hereby incorporated by reference). UNA is unlocked nucleic acid,typically where the C2-C3 C—C bond of the ribose has been removed,forming an unlocked “sugar” residue.

II.G. Oligomer Design

The oligomer of the invention can comprise a nucleotide sequence whichcomprises both nucleotides and nucleotide analogs, and can be in theform of a gapmer, blockmer, mixmer, headmer, tailmer, or totalmer.Examples of configurations of a gapmer, blockmer, mixmer, headmer,tailmer, or totalmer that can be used with the oligomer of the inventionare described in U.S. Patent Appl. Publ. No. 2012/0322851, which isincorporated by reference herein in its entirety.

A gapmer oligomer is an oligomer which comprises a contiguous stretch ofnucleotides which is capable of recruiting an RNase, such as RNaseH,such as a region of at least 7 DNA nucleotides, which is flanked both 5′and 3′ by regions of affinity enhancing 1-6 nucleotide analogs 5′ and 3′to the contiguous stretch of nucleotides which is capable of recruitingRNase.

A “headmer” is defined as an oligomer that comprises a region X and aregion Y that is contiguous thereto, with the 5′-most monomer of regionY linked to the 3′-most monomer of region X. Region X comprises acontiguous stretch of non-RNase recruiting nucleoside analogs and regionY comprises a contiguous stretch (such as at least 7 contiguousmonomers) of DNA monomers or nucleoside analog monomers recognizable andcleavable by the RNase.

A “tailmer” is defined as an oligomer that comprises a region X and aregion Y that is contiguous thereto, with the 5′-most monomer of regionY linked to the 3′-most monomer of the region X. Region X comprises acontiguous stretch (such as at least 7 contiguous monomers) of DNAmonomers or nucleoside analog monomers recognizable and cleavable by theRNase, and region X comprises a contiguous stretch of non-RNaserecruiting nucleoside analogs.

Other “chimeric” oligomers, called “mixmers”, consist of an alternatingcomposition of (i) DNA monomers or nucleoside analog monomersrecognizable and cleavable by RNase, and (ii) non-RNase recruitingnucleoside analog monomers.

A “totalmer” is a single stranded oligomer which only comprisesnon-naturally occurring nucleotides or nucleotide analogs.

In some embodiments, in addition to enhancing affinity of the oligomerfor the target region, some nucleoside analogs also mediate RNase (e.g.,RNaseH) binding and cleavage. Since α-L-LNA monomers recruit RNaseHactivity to a certain extent, in some embodiments, gap regions (e.g.,region B as referred to herein) of oligomers containing α-L-LNA monomersconsist of fewer monomers recognizable and cleavable by the RNaseH, andmore flexibility in the mixmer construction is introduced.

II.G.1. Gapmer Design

In one embodiment, the oligomer of the invention is a gapmer. A gapmeroligomer is an oligomer which comprises a contiguous stretch ofnucleotides which is capable of recruiting an RNase, such as RNaseH,such as a region of at least 7 DNA nucleotides, referred to herein in asregion B (B), wherein region B is flanked both 5′ and 3′ by regions ofaffinity enhancing nucleotide analogs, such as from 1-10 nucleotideanalogs 5′ and 3′ to the contiguous stretch of nucleotides which iscapable of recruiting RNase—these regions are referred to as regions A(A) and C (C) respectively.

In certain embodiments, the gapmer is an alternating flank gapmer,examples of which are discussed below. In certain embodiments, thealternating flank gapmer exhibits less off target binding than atraditional gapmer. In certain embodiments, the alternating flank gapmerhas better long term tolerability than a traditional gapmer.

An alternating flank gapmer can comprises a (poly)nucleotide sequence offormula (5′ to 3′), A-B-C, wherein: region A (A) (5′ region or a firstwing sequence) comprises at least one nucleotide analog, such as atleast one LNA unit, such as from 1-10 nucleotide analogs, such as LNAunits, and; region B (B) comprises at least seven consecutivenucleotides which are capable of recruiting RNase (when formed in aduplex with a complementary RNA molecule, such as the pre-mRNA or mRNAtarget), such as DNA nucleotides, and; region C (C) (3′region or asecond wing sequence) comprises at least one nucleotide analog, such asat least one LNA unit, such as from 1-10 nucleotide analogs, such as LNAunits; wherein regions A and C can include at any position in A and C1-2 insertions of DNA nucleotide regions (e.g., DNA gapmers), in whichthese DNA insertions can each be 1-3 DNA units long.

In certain other embodiments, the gapmer, e.g., an alternating flankgapmer, comprises a (poly)nucleotide sequence of formula (5′ to 3′),A-B-C, or optionally A-B-C-D or D-A-B-C, wherein: region A (A) (5′region) comprises at least one nucleotide analog, such as at least oneLNA unit, such as from 1-10 nucleotide analogs, such as LNA units, and;region B (B) comprises at least seven consecutive nucleotides which arecapable of recruiting RNase (when formed in a duplex with acomplementary RNA molecule, such as the mRNA target), such as DNAnucleotides, and; region C (C) (3′region) comprises at least onenucleotide analog, such as at least one LNA unit, such as from 1-10nucleotide analogs, such as LNA units, and; region D (D), when presentcomprises 1, 2 or 3 nucleotide units, such as DNA nucleotides.

In some embodiments, region A comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotide analogs, such as LNA units, such as from 2-5 nucleotideanalogs, such as 2-5 LNA units, such as 2-5 nucleotide analogs, such as3-5 LNA units; and/or region C consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 nucleotide analogs, such as LNA units, such as from 2-5 nucleotideanalogs, such as 2-5 LNA units, such as 2-5 nucleotide analogs, such as3-5 LNA units.

In some embodiments B comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, or 23 consecutive nucleotides which are capable ofrecruiting RNase, or from 8-14, or from 7-10, or from 7-9, such as 8,such as 9, such as 10, or such as 14 consecutive nucleotides which arecapable of recruiting RNase. In some embodiments region B comprises atleast seven DNA nucleotide unit, such as 7-23 DNA units, such as from7-20 DNA units, such as from 7-14 DNA units, such as from 8-14 DNAunits, such as 7, 8, 9, 10, 11, 12, 13, or 14 DNA units.

In some embodiments region A comprises 3, 4, or 5 nucleotide analogs,such as LNA, region B consists of 7, 8, 9, 10, 11, 12, 13, or 14 DNAunits, and region C consists of 3, 4, or 5 nucleotide analogs, such asLNA. Such designs include (A-B-C) 5-10-5, 3-14-3, 3-10-3, 3-10-4,4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, and4-7-3, and can further include region D, which can have one to 3nucleotide units, such as DNA units.

In some embodiments, the oligomer of the invention, e.g., an alternatingflank gapmer, has the formula of 5′-A-B-C-3′, wherein

-   (i) B is a contiguous sequence of 7 to 23 DNA units;-   (ii) A is a first wing sequence of 1 to 10 nucleotides, wherein the    first wing sequence comprises one or more nucleotide analogs and    optionally one or more DNA units (e.g., DNA gapmer) and wherein at    least one of the nucleotide analogs is located at the 5′ end of A;    and-   (iii) C is a second wing sequence of 1 to 10 nucleotides, wherein    the second wing sequence comprises one or more nucleotide analogs    and optionally one or more DNA units (e.g., DNA gapmer) and wherein    at least one of the nucleotide analogs is located at the 3′ end of    C.

In other embodiments, the oligomer, e.g., an alternating flank gapmer,has the formula of 5′-A-B-C-3′, wherein B is a contiguous sequence of 7to 23 DNA units, A is LmDnLoDpLq and C is Lm′Dn′Lo′Dp′Lq′ and wherein Lis a nucleotide analog; D is a DNA unit; m and q′ are 1 to 6 units; n,p, n′, and p′ are 0 to 2 units; and o, q, m′, and o′ are 0 to 5.

In some embodiments, the first wing sequence (A in the formula)comprises a combination of nucleotide analogs and DNA units selectedfrom (i) 1-9 nucleotide analogs and 1 DNA unit; (ii) 1-8 nucleotideanalogs and 1-2 DNA units; (iii) 1-7 nucleotide analogs and 1-3 DNAunits; (iv) 1-6 nucleotide analogs and 1-4 DNA units; (v) 1-5 nucleotideanalogs and 1-5 DNA units; (vi) 1-4 nucleotide analogs and 1-6 DNAunits; (vii) 1-3 nucleotide analogs and 1-7 DNA units; (viii) 1-2nucleotide analogs and 1-8 DNA units; and (ix) 1 nucleotide analog and1-9 DNA units.

In certain embodiments, the second wing sequence (C in the formula)comprises a combination of nucleotide analogs and DNA unit selected from(i) 1-9 nucleotide analogs and 1 DNA unit; (ii) 1-8 nucleotide analogsand 1-2 DNA units; (iii) 1-7 nucleotide analogs and 1-3 DNA units; (iv)1-6 nucleotide analogs and 1-4 DNA units; (v) 1-5 nucleotide analogs and1-5 DNA units; (vi) 1-4 nucleotide analogs and 1-6 DNA units; (vii) 1-3nucleotide analogs and 1-7 DNA units; (viii) 1-2 nucleotide analogs and1-8 DNA units; and (ix) 1 nucleotide analog and 1-9 DNA units.

In some embodiments, A in the oligomer formula has a sub-formulaselected from L, LL, LDL, LLL, LLDL, LDLL, LDDL, LLLL, LLLLL, LLLDL,LLDLL, LDLLL, LLDDL, LDDLL, LLDLD, LDLLD, LDDDL, LLLLLL, LLLLDL, LLLDLL,LLDLLL, LDLLLL, LLLDDL, LLDLDL, LLDDLL, LDDLLL, LDLLDL, LDLDLL, LDDDLL,LLDDDL, and LDLDLD, and C in the oligomer formula has a sub-formulaselected from L, LL, LDL, LLL, LLDL, LLLL, LDLL, LDDL, LLDD, LLLLL,LLLLD, LLLDL, LLDLL, LDLLL, LLDDL, LDDLL, LLDLD, LDLLD, LDDDL, LLLLLL,LLLLDL, LLLDLL, LLDLLL, LDLLLL, LLLDDL, LLDLDL, LLDDLL, LDDLLL, LDLLDL,LDLDLL, LDDDLL, LLDDDL, and LDLDLD.

In certain embodiments, the oligomer, e.g., an alternating flank gapmer,has the formula of 5′ A-B-C 3′, wherein B is a contiguous sequence of 7to 23 DNA units, A has a formula of LLDLL, LDLLL, or LLLDL and C has theformula of LLDLL or LDLDLL, and wherein L is an LNA unit and D is a DNAunit.

In other embodiments, the oligomers of the invention are alternatingflank gapmers having the formula of 5′ A-B-C 3′, wherein the oligomerhas 12 to 25 nucleotides in length, A is a first wing sequence havingthe formula of L_(m)d_(n)L_(o)d_(p)L_(q), C is a second wing sequencehaving the formula of L_(q′)d_(p′)L_(o′)d_(n′)L_(m), wherein each wingindependently has 1-17 nucleotides in length and is optionallyinterrupted by DNA spacers d_(n), d_(p), d_(n′) and d_(p), each of whichindependently has 0 to 3 DNA units, with each wing flanking an all DNAgap of 7 to 23 nucleotides;

wherein m and m′ are at least 1;

and n, n′, p and p′ are independently 0-3 units;

such that m+n+o+p+q=1-17; and independently m′+n′+o′+p′+q′=1-17;

or (m+n+o+p+q) and (m′+n′+o′+p′+q′) are independently 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17;

or B comprises a DNA gap of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22 or 23.

Further gapmer designs are disclosed in WO2004/046160, which is herebyincorporated by reference in its entirety. WO2008/113832 herebyincorporated by reference in its entirety, refers to ‘shortmer’ gapmeroligomers. In some embodiments, oligomers presented herein can be suchshortmer gapmers.

In some embodiments the oligomer, e.g., an alternating flank gapmer,comprises a contiguous nucleotide sequence of a total of 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 nucleotide units, wherein the contiguousnucleotide sequence is of formula (5′-3′), A-B-C, or optionally A-B-C-Dor D-A-B-C, wherein; A consists of 1, 2, 3, 4, or 5 nucleotide analogunits, such as LNA units; B consists of 7, 8, 9, 10, 11, 12, 13, or 14contiguous nucleotide units which are capable of recruiting RNase whenformed in a duplex with a complementary RNA molecule (such as a mRNAtarget); and C consists of 1, 2,3, 4, or 5 nucleotide analog units, suchas LNA units. When present, D consists of a single DNA unit.

In some embodiments A comprises 1 LNA unit. In some embodiments Acomprises 2 LNA units. In some embodiments A comprises 3 LNA units. Insome embodiments A comprises 4 LNA units. In some embodiments Acomprises 5 LNA units. In some embodiments C comprises 1 LNA unit. Insome embodiments C comprises 2 LNA units. In some embodiments Ccomprises 3 LNA units. In some embodiments C comprises 4 LNA units. Insome embodiments C comprises 5 LNA units. In some embodiments Bcomprises 7 nucleotide units. In some embodiments B comprises 8nucleotide units. In some embodiments B comprises 9 nucleotide units. Incertain embodiments, B comprises 10 nucleoside units. In certainembodiments, B comprises 11 nucleoside units. In certain embodiments, Bcomprises 12 nucleoside units. In certain embodiments, B comprises 13nucleoside units. In certain embodiments, B comprises 14 nucleosideunits. In certain embodiments, B comprises 7-23 DNA monomers. In someembodiments B comprises from 7-23 DNA units, such as 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 DNA units. In someembodiments B consists of DNA units. In some embodiments B comprises atleast one LNA unit which is in the alpha-L configuration, such as 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or23 LNA units in the alpha-L-configuration. In some embodiments Bcomprises at least one alpha-L-oxy LNA unit or wherein all the LNA unitsin the alpha-L-configuration are alpha-L-oxy LNA units. In someembodiments the number of nucleotides present in A-B-C are selected from(nucleotide analog units—region B-nucleotide analog units): 1-8-1,1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8-4,or; 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1, 4-9-1,1-9-4, or; 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, and 3-10-1. In someembodiments the number of nucleotides in A-B-C is selected from: 2-7-1,1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. In otherembodiments, the oligomer contains 10 DNA units in B, LDLLL in A (firstwing) and LLDLL in C (second wing). In yet other embodiments, theoligomer contains 9 DNA units in B, LDDLL in A, and LDLDLL in C. Instill other embodiments, the oligomer contains 10 DNA units in B, LLDLLin A, and LLDLL in C. In further embodiments, the oligomer contains 9DNA units in B, LLLLL in A, and LDDLL in C. In certain embodiments, eachof regions A and C comprises three LNA monomers, and region B consistsof 7, 8, 9, 10, 11, 12, 13, or 14 nucleoside monomers, for example, DNAmonomers. In some embodiments both A and C consist of two LNA unitseach, and B consists of 7, 8, or 9 nucleotide units, for example DNAunits. In various embodiments, other gapmer designs include those whereregions A and/or C consists of 3, 4, 5 or 6 nucleoside analogs, such asmonomers containing a 2′-O-methoxyethyl-ribose sugar (2′-MOE) ormonomers containing a 2′-fluoro-deoxyribose sugar, and region B consistsof 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23nucleosides, such as DNA monomers, where regions A-B-C have 3-8-3,3-9-3, 3-10-3, 5-10-5 or 4-12-4 monomers. Further gapmer designs aredisclosed in WO 2007/146511A2, hereby incorporated by reference in itsentirety.

In some embodiments, the alternating flank oligomer has at least 10contiguous nucleotides, comprising region A, region B, and region C(A-B-C), wherein region B comprises at least 5 consecutive nucleosideunits and is flanked at 5′ by region A of 1-8 contiguous nucleosideunits and at 3′ by region C of 1-8 contiguous nucleoside units, whereinregion B, when formed in a duplex with a complementary RNA, is capableof recruiting RNaseH, and wherein region A and region C are selectedfrom the group consisting of: (i) region A comprises a 5′ LNA nucleosideunit and a 3′ LNA nucleoside unit, and at least one DNA nucleoside unitbetween the 5′ LNA nucleoside unit and the 3′ LNA nucleoside unit, and,region C comprises at least two 3′ LNA nucleosides; or (ii) region Acomprises at least one 5′ LNA nucleoside and region C comprises a 5′ LNAnucleoside unit, at least two terminal 3′ LNA nucleoside units, and atleast one DNA nucleoside unit between the 5′ LNA nucleoside unit and the3′ LNA nucleoside units, and (iii) region A comprises a 5′ LNAnucleoside unit and a 3′ LNA nucleoside unit, and at least one DNAnucleoside unit between the 5′ LNA nucleoside unit and the 3′ LNAnucleoside unit; and region C comprises a 5′ LNA nucleoside unit, atleast two terminal 3′ LNA nucleoside units, and at least one DNAnucleoside unit between the 5′ LNA nucleoside unit and the 3′ LNAnucleoside units.

In some embodiments, region A or region C comprises 1, 2, or 3 DNAnucleoside units. In other embodiments, region A and region C comprise1, 2, or 3 DNA nucleoside units. In yet other embodiments, region Bcomprises at least five consecutive DNA nucleoside units. In certainembodiments, region B comprises 6, 7, 8, 9, 10, 11, 12, 13 or 14consecutive DNA nucleoside units. In some embodiments, region B is 8, 910, 11, or 12 nucleotides in length. In other embodiments, region Acomprises two 5′ terminal LNA nucleoside units. In some embodiments,region A has formula 5′[LNA]₁₋₃[DNA]₁₋₃[LNA]₁₋₃, or5′[LNA]₁₋₂[DNA]₁₋₂[LNA]₁₋₂[DNA]₁₋₂[LNA]₁₋₂. In other embodiments, regionC has formula [LNA]₁₋₃[DNA]₁₋₃[LNA]₂₋₃3′, or[LNA]₁₋₂[DNA]₁₋₂[LNA]₁₋₂[DNA]₁₋₂[LNA]₂₋₃3′ In yet other embodiments,region A has formula 5′[LNA]₁₋₃[DNA]₁₋₃[LNA]₁₋₃, or5′[LNA]₁₋₂[DNA]₂[LNA]₁₋₂[DNA]₁₋₂[LNA]₁₋₂, and region C comprises 2, 3, 4or 5 consecutive LNA nucleoside units. In some embodiments, region C hasformula [LNA]₁₋₃[DNA]₁₋₃[LNA]₂₋₃3′ or[LNA]₁₋₂[DNA]₁₋₂[LNA]₁₋₂[DNA]₁₋₂[LNA]₂₋₃3′, and region A comprises 1, 2,3, 4 or 5 consecutive LNA nucleoside units. In still other embodiments,region A has a sequence of LNA and DNA nucleosides, 5′-3′ selected fromthe group consisting of L, LL, LDL, LLL, LLDL, LDLL, LDDL, LLLL, LLLLL,LLLDL, LLDLL, LDLLL, LLDDL, LDDLL, LLDLD, LDLLD, LDDDL, LLLLLL, LLLLDL,LLLDLL, LLDLLL, LDLLLL, LLLDDL, LLDLDL, LLDDLL, LDDLLL, LDLLDL, LDLDLL,LDDDLL, LLDDDL, and LDLDLD, wherein L represents a LNA nucleoside, and Drepresents a DNA nucleoside. In yet other embodiments, region C has asequence of LNA and DNA nucleosides, 5′-3′ selected from the groupconsisting of LL, LLL, LLLL, LDLL, LLLLL, LLDLL, LDLLL, LDDLL, LDDLLL,LLDDLL, LDLDLL, LDDDLL, LDLDDLL, LDDLDLL, LDDDLLL, and LLDLDLL. In afurther embodiment, region A has a sequence of LNA and DNA nucleosides,5′-3′ selected from the group consisting of LDL, LLDL, LDLL, LDDL,LLLDL, LLDLL, LDLLL, LLDDL, LDDLL, LLDLD, LDLLD, LDDDL, LLLLDL, LLLDLL,LLDLLL, LDLLLL, LLLDDL, LLDLDL, LLDDLL, LDDLLL, LDLLDL, LDLDLL, LDDDLL,LLDDDL, and LDLDLD, and region C has a sequence of LNA and DNAnucleosides, 5′-3′ selected from the group consisting of LDLL, LLLLL,LLDLL, LDLLL, LDDLL, LDDLLL, LLDDLL, LDLDLL, LDDDLL, LDLDDLL, LDDLDLL,LDDDLLL, and LLDLDLL.

In certain embodiments, the alternating flank oligomer has contiguousnucleotides comprising a sequence of nucleosides, 5′-3′, selected fromthe group consisting of LLDDDLLDDDDDDDDLL, LDLLDLDDDDDDDDDLL,LLLDDDDDDDDDDLDLL, LLLDDDDDDDDDLDDLL, LLLDDDDDDDDLDDDLL,LLLDDDDDDDDLDLDLL, LLLDLDDDDDDDDDLLL, LLLDLDDDDDDDDLDLL,LLLLDDDDDDDDDLDLL, LLLLDDDDDDDDLDDLL, LLLDDDLDDDDDDDDLL,LLLDDLDDDDDDDDDLL, LLLDDLLDDDDDDDDLL, LLLDDLLDDDDDDDLLL,LLLLLDDDDDDDLDDLL, LDLLLDDDDDDDDDDLL, LDLLLDDDDDDDLDDLL,LDLLLLDDDDDDDDDLL, LLDLLLDDDDDDDDDLL, LLLDLDDDDDDDDDDLL,LLLDLDDDDDDDLDDLL, LLLDLLDDDDDDDDDLL, LLLLDDDDDDDLDDDLL,LLLLLDDDDDDDDDLDLL, LLLLDDDDDDDDDDLDLL, LLLDDDDDDDDDDDLDLL,LLDLDDDDDDDDDDLDLL, LDLLLDDDDDDDDDLDLL, LLLDDDDDDDDDDLDDLL,LLLDDDDDDDDDLDDDLL, LLLDDDDDDDDLDLDDLL, LLLLDDDDDDDDDLDDLL,LLLLDDDDDDDDDLDLLL, LLLLDDDDDDDDLDDDLL, LLLLDDDDDDDDLDDLLL,LLLLDDDDDDDDLDLDLL, LLLLDDDDDDDLDDLDLL, LLLLDDDDDDDLDLDDLL,LLDLLDDDDDDDDDDDLL, LLDLLLDDDDDDDDLDLL, LLLDLDDDDDDDDDDDLL,LLLDLDDDDDDDDDLDLL, LLLDLDDDDDDDDLDDLL, LLLDLDDDDDDDLDLDLL,LLLLDDDDDDDDDLLDLL, LLLLLDDDDDDDDDLDLLL, LLLLLDDDDDDDDDLDDLL,LLLLDDDDDDDDDDLLDLL, LLLLDDDDDDDDDDLDLLL, LLLLDDDDDDDDDDLDDLL,LLLDDDDDDDDDDDLLDLL, LLLDDDDDDDDDDDLDLLL, LLLLLDDDDDDDDDLLDLL,LLLDDDDDDDDDDDLDDLL, LLDLLDDDDDDDDDLDDLL, LLLDLDDDDDDDDDDLDLL,LLLDLDDDDDDDDDLDDLL, LLLLDDDDDDDDDLDLDLL, LLLLDDDDDDDDLLDLDLL,LDLLLDDDDDDDDDDLLDLL, LLDLLDDDDDDDDDDLLDLL, LLDLDDDDDDDDDDDDLLLL,LLDDLDDDDDDDDDDDLLLL, LLLDLDDDDDDDDDDDLLLL, LLDLDDDDDDDDDDDDDLLL,LLDLLDDDDDDDDDDDLLLL, LLDDLDDDDDDDDDDDDLLL, LLLDDDDDDDDDDDLDDLLL,LLLDLDDDDDDDDDDDDLLL, LLDLLDDDDDDDDDDDDLLL, LLLLDDDDDDDDDDDLLDLL,LLLLDDDDDDDDDDLLDDLL, LLLDDLDDDDDDDDDLDLLL, LLDDLDLDDDDDDDDDLLLL,LLDDLLDDDDDDDDDLDLLL, LLLDLDDDDDDDDDLDLDLL, LLDLLDDDDDDDDDLDDLLL,LLLDLDDDDDDDDDDLDLLL, LLDLDDLDDDDDDDDDLLLL, LLLLDDDDDDDDDLDLDDLL,LLLDLDDDDDDDDDLDDLLL, LLDLDLDDDDDDDDDDLLLL, LLDLLDDDDDDDDDDLDLLL,LLDLDLDDDDDDDDDLLDLL, LLDDLLDDDDDDDDDLLDLL, LLLLDDDDDDDDDLDDLDLL,LLLDDLDDDDDDDDDLLDLL, LLDLLDDDDDDDDDLLDDLL, LLDLDLDDDDDDDDDLDLLL,LLLDLDDDDDDDDDLLDDLL, LLDDLLDDDDDDDDDDLLLL, LLDLLDDDDDDDDDLDLDLL,LLLLDDDDDDDDDDLDDLLL, LLLDDLDDDDDDDDDDLLLL, LLLDLDDDDDDDDDDLLDLL,LLLLDDDDDDDDDDLDLDLL, LLLLDDDDDDDDDDDLDLLL, and LLDDLLDDDDDDDDDDLDLL;wherein L represents a LNA nucleoside, and D represents a DNAnucleoside. In other embodiments, the LNA nucleoside is beta-D-oxy LNA.

In yet other embodiments, an alternating flank oligomer has contiguousnucleotides comprising an alternating sequence of LNA and DNA nucleosideunits, 5′-3′, selected from the group consisting of: 2-3-2-8-2,1-1-2-1-1-9-2, 3-10-1-1-2, 3-9-1-2-2, 3-8-1-3-2, 3-8-1-1-1-1-2,3-1-1-9-3, 3-1-1-8-1-1-2, 4-9-1-1-2, 4-8-1-2- 2, 3-3-1-8-2, 3-2-1-9-2,3-2-2-8-2, 3-2-2-7-3, 5-7-1-2-2, 1-1-3-10-2, 1-1-3-7-1-2-2, 1-1-4-9-2,2-1-3-9-2, 3-1-1-10-2, 3-1-1-7-1-2-2, 3-1-2-9-2, 4-7-1-3-2, 5-9-1-1-2,4-10-1-1-2, 3-11-1-1-2, 2-1-1-10-1-1-2, 1-1-3-9-1-1-2, 3-10-1-2-2,3-9-1-3-2, 3-8-1-1-1-2-2, 4-9-1-2-2, 4-9-1-1-3, 4-8-1-3-2, 4-8-1-2-3,4-8-1-1-1-1-2, 4-7-1-2-1-1-2, 4-7-1-1-1-2-2, 2-1-2-11-2, 2-1-3-8-1-1-2,3-1-1-11-2, 3-1-1-9-1-1-2, 3-1-1-8-1-2-2, 3-1-1-7-1-1-1-1-2, 4-9-2-1-2,4- 7-1-3-3, 5-9-1-1-3, 5-9-1-2-2, 4-10-2-1-2, 4-10-1-1-3, 4-10-1-2-2,3-11-2-1-2, 3-11-1-1-3, 5-9- 2-1-2, 3-11-1-2-2, 2-1-2-9-1-2-2,3-1-1-10-1-1-2, 3-1-1-9-1-2-2, 4-9-1-1-1-1-2, 4-8- 2-1-1-1-2,1-1-3-10-2-1-2, 2-1-2-10-2-1-2, 2-1-1-12-4, 2-2-1-11-4, 3-1-1-11-4,2-1-1-13-3, 2- 1-2-11-4, 2-2-1-12-3, 3-11-1-2-3, 3-1-1-12-3, 2-1-2-12-3,4-11-2-1-2, 4-10-2-2-2, 3-2-1- 9-1-1-3, 2-2-1-1-1-9-4, 2-2-2-9-1-1-3,3-1-1-9-1-1-1-1-2, 2-1-2-9-1-2-3, 3-1-1-10-1-1-3, 2-1-1-2-1-9-4,4-9-1-1-1-2-2, 3-1-1-9-1-2-3, 2-1-1-1-1-10-4, 2-1-2-10-1-1-3,2-1-1-1-1-9-2-1-2, 2-2-2-9-2-1-2, 4-9-1-2-1-1-2, 3-2-1-9-2-1-2,2-1-2-9-2-2-2, 2-1-1-1-1-9-1-1-3, 3-1-1-9-2-2-2, 2-2-2-10-4,2-1-2-9-1-1-1-1-2, 4-10-1-2-3, 3-2-1-10-4, 3-1-1-10-2-1-2,4-10-1-1-1-1-2, 4-11-1-1-3, and 2-2-2-10-1-1-2; wherein the firstnumeral represents an number of LNA units, the next a number of DNAunits, and alternating LNA and DNA regions thereafter.

In other embodiments, the oligomers of the invention has the designdescribed in FIGS. 2, 3, 6, 7, 16A, 16B, 20A, or 20B.

II.H. Internucleotide Linkages

The monomers of the oligomers described herein are coupled together vialinkage groups.

Suitably, each monomer is linked to the 3′ adjacent monomer via alinkage group.

The person having ordinary skill in the art would understand that, inthe context of the present invention, the 5′ monomer at the end of anoligomer does not comprise a 5′ linkage group, although it may or maynot comprise a 5′ terminal group.

The terms “linkage group” or “internucleotide linkage” are intended tomean a group capable of covalently coupling together two nucleotides.Specific and preferred examples include phosphate groups andphosphorothioate groups.

The nucleotides of the oligomer of the invention or contiguousnucleotides sequence thereof are coupled together via linkage groups.Suitably each nucleotide is linked to the 3′ adjacent nucleotide via alinkage group.

Suitable internucleotide linkages include those listed withinWO2007/031091, for example the internucleotide linkages listed on thefirst paragraph of page 34 of WO2007/031091 (hereby incorporated byreference in its entirety).

Examples of suitable internucleotide linkages that can be used with theinvention include phosphodiester linkage, a phosphotriester linkage, amethylphosphonate linkage, a phosphoramidate linkage, a phosphorothioatelinkage, and combinations thereof.

It is, in some embodiments, preferred to modify the internucleotidelinkage from its normal phosphodiester to one that is more resistant tonuclease attack, such as phosphorothioate or boranophosphate—these two,being cleavable by RNaseH, also allow that route of antisense inhibitionin reducing the expression of the target gene.

Suitable sulphur (S) containing internucleotide linkages as providedherein may be preferred. Phosphorothioate internucleotide linkages arealso preferred, particularly for the gap region (B) of gapmers.Phosphorothioate linkages can also be used for the flanking regions (Aand C, and for linking A or C to D, and within region D, asappropriate).

Regions A, B and C, can, however, comprise internucleotide linkagesother than phosphorothioate, such as phosphodiester linkages,particularly, for instance when the use of nucleotide analogs protectsthe internucleotide linkages within regions A and C from endo-nucleasedegradation—such as when regions A and C comprise LNA nucleotides.

The internucleotide linkages in the oligomer can be phosphodiester,phosphorothioate or boranophosphate so as to allow RNaseH cleavage oftargeted RNA. Phosphorothioate is preferred, for improved nucleaseresistance and other reasons, such as ease of manufacture.

In one aspect of the oligomer of the invention, the nucleotides and/ornucleotide analogs are linked to each other by means of phosphorothioategroups.

It is recognized that the inclusion of phosphodiester linkages, such asone or two linkages, into an otherwise phosphorothioate oligomer,particularly between or adjacent to nucleotide analog units (typicallyin region A and or C) can modify the bioavailability and/orbio-distribution of an oligomer—see WO2008/113832, hereby incorporatedby reference.

In some embodiments, such as the embodiments referred to above, wheresuitable and not specifically indicated, all remaining linkage groupsare either phosphodiester or phosphorothioate, or a mixture thereof.

In some embodiments all the internucleotide linkage groups arephosphorothioate.

When referring to specific gapmer oligonucleotide sequences, such asthose provided herein it will be understood that, in variousembodiments, when the linkages are phosphorothioate linkages,alternative linkages, such as those disclosed herein can be used, forexample phosphate (phosphodiester) linkages can be used, particularlyfor linkages between nucleotide analogs, such as LNA, units. Likewise,when referring to specific gapmer oligonucleotide sequences, such asthose provided herein, when the C residues are annotated as 5′methylmodified cytosine, in various embodiments, one or more of the Cs presentin the oligomer can be unmodified C residues.

US Publication No. 2011/0130441, which was published Jun. 2, 2011 and isincorporated by reference herein in its entirety, refers to oligomericcompounds having at least one bicyclic nucleoside attached to the 3′ or5′ termini by a neutral internucleoside linkage. The oligomers of theinvention can therefore have at least one bicyclic nucleoside attachedto the 3′ or 5′ termini by a neutral internucleoside linkage, such asone or more phosphotriester, methylphosphonate, MMI, amide-3, formacetalor thioformacetal. The remaining linkages can be phosphorothioate.

In some embodiments, the oligomers of the invention have internucleotidelinkages described in FIG. 2, 16B, or 20B. As used herein, e.g., FIGS.2, 16B, or 20B, phosphorothioate linkages are indicated as “s”, andphosphorodiester linkages are indicated by the absence of “s.”

In some embodiments, the internucleotide linkages are combinations ofphosphorothioate linkages and phosphodiester linkages. Non-limitingexamples of combination linkages are shown in ASO-002623, ASO-002667,ASO-002674, ASO-002631, ASO-002639, and ASO-002624.

III. Conjugates

In the context the term “conjugate” is intended to indicate aheterogeneous molecule formed by the covalent or non-covalent attachment(“conjugation”) of the oligomer as described herein to one or morenon-nucleotide, or non-polynucleotide moieties. Examples ofnon-nucleotide or non-polynucleotide moieties include macromolecularagents such as proteins, fatty acid chains, sugar residues,glycoproteins, polymers, or combinations thereof. Typically proteins canbe antibodies for a target protein. In some embodiments, typicalpolymers are polyethylene glycol.

Therefore, in various embodiments, the oligomer of the inventioncomprises both a polynucleotide region which typically consists of acontiguous sequence of nucleotides, and a further non-nucleotide region.When referring to the oligomer of the invention comprising a contiguousnucleotide sequence, the compound can comprise non-nucleotidecomponents, such as a conjugate component.

The invention also provides for a conjugate comprising the oligomeraccording to the invention as herein described, and at least onenon-nucleotide or non-polynucleotide moiety covalently attached to theoligomer. Therefore, in various embodiments where the oligomer of theinvention comprises a specified nucleic acid or nucleotide sequence, asherein disclosed, the compound can also comprise at least onenon-nucleotide or non-polynucleotide moiety (e.g., not comprising one ormore nucleotides or nucleotide analogs) covalently attached to theoligomer.

Conjugation (to a conjugate moiety) can enhance the activity, cellulardistribution or cellular uptake of the oligomer of the invention. Suchmoieties include, but are not limited to, antibodies, polypeptides,lipid moieties such as a cholesterol moiety, cholic acid, a thioether.

The oligomers of the invention can also be conjugated to active drugsubstances, for example, aspirin, ibuprofen, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

In certain embodiments the conjugated moiety is a sterol, such ascholesterol.

III.A. Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer ofthe invention that is covalently linked (i.e., functionalized) to atleast one functional moiety that permits covalent linkage of theoligomer to one or more conjugated moieties, i.e., moieties that are notthemselves nucleic acids or monomers, to form the conjugates hereindescribed. Typically, a functional moiety will comprise a chemical groupthat is capable of covalently bonding to the oligomer via, e.g., a3′-hydroxyl group or the exocyclic NH₂ group of the adenine base, aspacer that can be hydrophilic and a terminal group that is capable ofbinding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxylgroup).

In some embodiments, this terminal group is not protected, e.g., is anNH₂ group. In other embodiments, the terminal group is protected, forexample, by any suitable protecting group such as those described in“Protective Groups in Organic Synthesis” by Theodora W Greene and PeterG M Wuts, 3rd edition (John Wiley & Sons, 1999).

In some embodiments, oligomers of the invention are functionalized atthe 5′ end in order to allow covalent attachment of the conjugatedmoiety to the 5′ end of the oligomer. In other embodiments, oligomers ofthe invention can be functionalized at the 3′ end. In still otherembodiments, oligomers of the invention can be functionalized along thebackbone or on the heterocyclic base moiety. In yet other embodiments,oligomers of the invention can be functionalized at more than oneposition independently selected from the 5′ end, the 3′ end, thebackbone and the base.

In some embodiments, activated oligomers of the invention aresynthesized by incorporating during the synthesis one or more monomersthat is covalently attached to a functional moiety. In otherembodiments, activated oligomers of the invention are synthesized withmonomers that have not been functionalized, and the oligomer isfunctionalized upon completion of synthesis.

IV. Pharmaceutical Compositions and Administration Routes

The oligomer of the invention can be used in pharmaceutical formulationsand compositions. Suitably, such compositions comprise apharmaceutically acceptable diluent, carrier, salt or adjuvant.

The oligomer of the invention can be included in a unit formulation suchas in a pharmaceutically acceptable carrier or diluent in an amountsufficient to deliver to a patient a therapeutically effective amountwithout causing serious side effects in the treated patient. However, insome forms of therapy, serious side effects may be acceptable in termsof ensuring a positive outcome to the therapeutic treatment.

The formulated drug may comprise pharmaceutically acceptable bindingagents and adjuvants. Capsules, tablets, or pills can contain forexample the following compounds: microcrystalline cellulose, gum orgelatin as binders; starch or lactose as excipients; stearates aslubricants; various sweetening or flavoring agents. For capsules thedosage unit may contain a liquid carrier like fatty oils. Likewisecoatings of sugar or enteric agents may be part of the dosage unit. Theoligonucleotide formulations can also be emulsions of the activepharmaceutical ingredients and a lipid forming a micellular emulsion.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be (a) oral (b) pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, (c) topical including epidermal, transdermal,ophthalmic and to mucous membranes including vaginal and rectaldelivery; or (d) parenteral including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion; orintracranial, e.g., intrathecal, intra-cerebroventricular, orintraventricular, administration. In one embodiment the oligomer isadministered IV, IP, orally, topically or as a bolus injection oradministered directly in to the target organ. In another embodiment, theoligomer is administered intrathecal or intra-cerebroventricular as abolus injection.

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, sprays, suppositories, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Examples of topical formulationsinclude those in which the oligomer of the invention are in admixturewith a topical delivery agent such as lipids, liposomes, fatty acids,fatty acid esters, steroids, chelating agents and surfactants.Compositions and formulations for oral administration include but arenot limited to powders or granules, microparticulates, nanoparticulates,suspensions or solutions in water or non-aqueous media, capsules, gelcapsules, sachets, tablets or minitablets. Compositions and formulationsfor parenteral, intrathecal, intra-cerebroventricular, orintraventricular administration can include sterile aqueous solutionswhich can also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Delivery ofdrug to the target tissue can be enhanced by carrier-mediated deliveryincluding, but not limited to, cationic liposomes, cyclodextrins,porphyrin derivatives, branched chain dendrimers, polyethyleniminepolymers, nanoparticles and microspheres (Dass C R. J Pharm Pharmacol2002; 54(0:3-27).

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

For parenteral, subcutaneous, intradermal or topical administration theformulation can include a sterile diluent, buffers, regulators oftonicity and antibacterials. The active oligomers can be prepared withcarriers that protect against degradation or immediate elimination fromthe body, including implants or microcapsules with controlled releaseproperties. For intravenous administration the carriers can bephysiological saline or phosphate buffered saline. InternationalPublication No. WO2007/031091 (A2), published Mar. 22, 2007, furtherprovides suitable pharmaceutically acceptable diluent, carrier andadjuvants—which are hereby incorporated by reference.

V. Diagnostics

This disclosure further provides a diagnostic method useful duringdiagnosis of Tau related diseases, e.g., a tauopathy. Non-limitingexamples of tauopathy include, but are not limited to, Alzheimer'sdisease, progressive supranuclear palsy, dementia pugilistica (chronictraumatic encephalopathy), frontal temporal dementia, parkinsonismlinked to chromosome 17, Lytico-Bodig disease (Parkinson-dementiacomplex of Guam), Tangle-predominant dementia, ganglioglioma,gangliocytoma, meningioangiomatosis, subacute sclerosingpanencephalitis, lead encephalopathy, tuberous sclerosis,Hallervorden-Spatz disease, Pick's disease, corticobasal ganglionicdegeneration, argyrophilic grain disease, corticobasal degeneration,lipofuscinosis, frontotemporal dementia, supranuclear palsy, orfrontotemporal lobar degeneration.

The oligomers of the invention can be used to measure expression of Tautranscript in a tissue or body fluid from an individual and comparingthe measured expression level with a standard Tau transcript expressionlevel in normal tissue or body fluid, whereby an increase in theexpression level compared to the standard is indicative of a disordertreatable by an oligomer of the invention.

The oligomer of the invention can be used to assay Tau transcript levelsin a biological sample using any methods known to those of skill in theart. (Touboul et. al., Anticancer Res. (2002) 22 (6A): 3349-56; Verjoutet. al., Mutat. Res. (2000) 640: 127-38); Stowe et. al., J. Virol.Methods (1998) 75 (1): 93-91).

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source of cellspotentially expressing Tau transcript. Methods for obtaining tissuebiopsies and body fluids from mammals are well known in the art.

VI. Kits Comprising Oligomers

This disclosure further provides kits that comprise an oligomer of theinvention described herein and that can be used to perform the methodsdescribed herein. In certain embodiments, a kit comprises at least oneoligomer in one or more containers. In some embodiments, the kitscontain all of the components necessary and/or sufficient to perform adetection assay, including all controls, directions for performingassays, and any necessary software for analysis and presentation ofresults. One skilled in the art will readily recognize that thedisclosed oligomer can be readily incorporated into one of theestablished kit formats which are well known in the art.

VII. Methods of Using

The oligomers of the invention can be utilized as research reagents for,for example, diagnostics, therapeutics and prophylaxis.

In research, such oligomers can be used to specifically inhibit thesynthesis of Tau protein (typically by degrading or inhibiting the mRNAand thereby prevent protein formation) in cells and experimental animalsthereby facilitating functional analysis of the target or an appraisalof its usefulness as a target for therapeutic intervention. Furtherprovided are methods of down-regulating the expression of MAPT mRNAand/or Tau protein in cells or tissues comprising contacting the cellsor tissues, in vitro or in vivo, with an effective amount of one or moreof the oligomers, conjugates or compositions of the invention.

In diagnostics the oligomers can be used to detect and quantitate MAPTtranscript expression in cell and tissues by northern blotting, in-situhybridization or similar techniques.

For therapeutics, an animal or a human, suspected of having a disease ordisorder, which can be treated by modulating the expression of MAPTtranscript and/or Tau protein is treated by administering oligomericcompounds in accordance with this invention. Further provided aremethods of treating a mammal, such as treating a human, suspected ofhaving or being prone to a disease or condition, associated withexpression ofMAPT transcript and/or Tau protein by administering atherapeutically or prophylactically effective amount of one or more ofthe oligomers or compositions of the invention. The oligomer, aconjugate or a pharmaceutical composition according to the invention istypically administered in an effective amount. In some embodiments, theoligomer or conjugate of the invention is used in therapy.

The invention further provides for an oligomer according to theinvention, for use for the treatment of one or more of the diseasesreferred to herein, such as a disease selected from Alzheimer's disease,progressive supranuclear palsy, Down syndrome, dementia pugilistica(chronic traumatic encephalopathy and other traumatic brain injury),frontal temportal dementia, frontal temporal dementia with parkinsonismlinked to chromosome 17 (FTDP-17), Lytico-Bodig disease(Parkinson-dementia complex of Guam), Tangle-predominant dementia,ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosingpanencephalitis, lead encephalopathy, Hemimegalencephaly, tuberoussclerosis, Hallervorden-Spatz disease, Pick's disease, corticobasalganglionic degeneration, argyrophilic grain disease, corticobasaldegeneration, lipofuscinosis, frontotemporal dementia, supranuclearpalsy, and frontotemporal lobar degeneration (reviewed in Frost et.al.,Trends Cell Biol (2015) 25: 216-53; Dyment et. al., Neurobiol. Aging(2014) September 6: S0197-4580; Moussaud et. al., Mol. Neurodeg (2014)29:43 Ross et. al., South Med. J. (2014) 107: 715-21). In addition, theinvention provides for oligomer use for the treatment diseases of brainnetwork dysfunction including all forms of epilepsy and depression(Inoue et. al., Epilepsy (2012) 102: 8-12; Xi et. al., Med Hypotheses(2011) 76: 897-900; Hou et. al., Can. J. Psychiatry (2004) 3: 164-71).The invention further provides for a method for treating tauopathies,the method comprising administering an effective amount of one or moreoligomers, conjugates, or pharmaceutical compositions thereof to ananimal in need thereof (such as a patient in need thereof).

In certain embodiments, the disease, disorder, or condition isassociated with overexpression of MAPT gene transcript and/or Tauprotein.

The invention also provides for methods of inhibiting (e.g., byreducing) the expression of MAPT gene transcript and/or Tau protein in acell or a tissue, the method comprising contacting the cell or tissue,in vitro or in vivo, with an effective amount of one or more oligomers,conjugates, or pharmaceutical compositions thereof, of the invention toaffect degradation of expression of MAPT gene transcript therebyreducing Tau protein.

The invention also provides for the use of the oligomer or conjugate ofthe invention as described for the manufacture of a medicament for thetreatment of a disorder as referred to herein, or for a method of thetreatment of as a disorder as referred to herein.

The invention further provides for a method for inhibiting Tau proteinin a cell which is expressing Tau comprising administering an oligomeror a conjugate according to the invention to the cell so as to affectthe inhibition of Tau protein in the cell.

The invention includes a method of reducing, ameliorating, preventing,or treating neuronal hyperexcitability in a subject in need thereofcomprising administering an oligomer or a conjugate according to theinvention.

The invention also provides for a method for treating a disorder asreferred to herein the method comprising administering an oligomer or aconjugate according to the invention as herein described and/or apharmaceutical composition according to the invention to a patient inneed thereof.

The oligomers and other compositions according to the invention can beused for the treatment of conditions associated with over expression orexpression of mutated version of Tau protein.

The invention provides for the oligomer or the conjugate according toinvention, for use as a medicament, such as for the treatment oftauopathies. In some embodiments the tauopathy is a disease selectedfrom Alzheimer's disease, progressive supranuclear palsy, dementiapugilistica (chronic traumatic encephalopathy), frontal temporaldementia, frontal temporal dementia with parkinsonism linked tochromosome 17, Lytico-Bodig disease (Parkinson-dementia complex ofGuam), Down syndrome, Hemimegalencephaly, Tangle-predominant dementia,ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosingpanencephalitis, lead encephalopathy, tuberous sclerosis,Hallervorden-Spatz disease, Pick's disease, corticobasal ganglionicdegeneration, argyrophilic grain disease, corticobasal degeneration,lipofuscinosis, frontotemporal dementia, supranuclear palsy, andfrontotemporal lobar degeneration (reviewed in Frost et al, Trends CellBiol (2015) 25: 216-53; Thom et al., Brain (2011) 134:2969-81; Zheng etal., Mol. Neurobiol. (2014) 49: 1532-9).

The invention provides for the oligomer or the conjugate according toinvention, for use as a medicament, such as for the treatment of seizuredisorders (Dyment et. al., Neurobiol. Aging (2014) September 6S0197-4580; Inoue et. al., Epilepsy (2012) 102:8-12; Gheyera et. al.,Ann Neurol (2014-76: 443-56). In some embodiments, the seizure disorderis selected from epilepsy, juvenile myoclonic epilepsy, reflex epilepsy,benign focal epilepsy of childhood (BFEC), generalized epilepsy withfebrile seizures plus (GEFS+), migrating partial seizures in infancy(MPSI), Mendelian epilepsy syndromes, infantile convulsions, infantilespasms, severe myoclonic epilepsy of infancy (SMEI or Dravet syndrome),Juvenile myoclonic epilepsy (JME or Janz syndrome), Angelman syndrome,Rett syndrome, epilepsy in fragile X syndrome, choreoathetosis (ICCA)syndrome, injury-associated seizures, brain injury, brain strokes,meningitis, and febrile seizures. In certain embodiments, the epilepsyis benign familial infantile epilepsy (BFIE).

In other embodiments, the seizure disorder is selected from idiopathicgeneralized epilepsy, idiopathic partial epilepsy, symptomaticgeneralized epilepsy, or symptomatic partial epilepsy. In someembodiments, the seizure disorder is idiopathic epilepsy selected fromchildhood absence epilepsy, juvenile myoclonic epilepsy, epilepsy withgrand mal seizures on awakening others, benign focal epilepsy ofchildhood. In certain embodiments, the seizure disorder is symptomaticepilepsy selected from West syndrome, Lennox-Gastaut syndrome, temporallobe epilepsy, or frontal lobe epilepsy. In other embodiments, theseizure disorder is idiopathic generalized epilepsy selected frommyoclonic seizures (sudden and very short duration jerking of theextremities), absence seizures (staring spells), or generalizedtonic-clonic seizures (grand mal seizures). In still other embodiments,the seizure disorder is idiopathic partial epilepsy including benignfocal epilepsy of childhood (BFEC).

The invention also provides for the oligomer or the conjugate accordingto the invention, for use as a medicament, such as for the treatment ofmovement disorders. In some embodiments, the movement disorder isselected from Akathisia (inability to sit still), Akinesia (lack ofmovement), Associated Movements (Mirror Movements or HomolateralSynkinesis), Athetosis (contorted torsion or twisting), Ataxia (grosslack of coordination of muscle movements), Ballismus (violentinvoluntary rapid and irregular movements), Hemiballismus (affectingonly one side of the body), Bradykinesia (slow movement), Cerebralpalsy, Chorea (rapid, involuntary movement), Sydenham's chorea,Rheumatic chorea, Huntington's disease, Dyskinesia (abnormal,involuntary movement), Tardive dyskinesia, Dystonia (sustained torsion),Dystonia muscularum, Blepharospasm, Writer's cramp, Spasmodictorticollis (twisting of head and neck), Dopamine-responsive dystonia(hereditary progressive dystonia with diurnal fluctuation or Segawa'sdisease), Essential tremor, Geniospasm (episodic involuntary up and downmovements of the chin and lower lip), Myoclonus (brief, involuntarytwitching of a muscle or a group of muscles), Metabolic GeneralUnwellness Movement Syndrome (MGUMS), Mirror movement disorder(involuntary movements on one side of the body mirroring voluntarymovements of the other side), Parkinson's disease, Paroxysmalkinesigenic dyskinesia, Restless Legs Syndrome RLS (WittMaack-Ekbomsdisease), Spasms (contractions), Stereotypic movement disorder,Stereotypy (repetition), Tic disorders (involuntary, compulsive,repetitive, stereotyped), Tourette's syndrome, Tremor (oscillations),Rest tremor (4-8 Hz), Postural tremor, Kinetic tremor, Essential tremor(6-8 Hz variable amplitude), Cerebellar tremor (6-8 Hz variableamplitude), Parkinsonian tremors (4-8 Hz variable amplitude),Physiological tremor (10-12 Hz low amplitude), Wilson's disease, andtics.

The invention further provides use of an oligomer of the invention inthe manufacture of a medicament for the treatment of a disease, disorderor condition as referred to herein. In some embodiments, the oligomer orconjugate of the invention is used for the manufacture of a medicamentfor the treatment of a tauopathy, a seizure disorder, or a combinationthereof.

Generally stated, one aspect of the invention is directed to a method oftreating a mammal suffering from or susceptible to conditions associatedwith abnormal levels of Tau i.e., a tauopathy), comprising administeringto the mammal and therapeutically effective amount of an oligomertargeted to MAPT transcript that comprises one or more LNA units. Theoligomer, a conjugate or a pharmaceutical composition according to theinvention is typically administered in an effective amount.

The disease or disorder, as referred to herein, can, in some embodimentsbe associated with a mutation in the MAPT gene or a gene whose proteinproduct is associated with or interacts with Tau protein. Therefore, insome embodiments, the target mRNA is a mutated form of the MAPTsequence.

An interesting aspect of the invention is directed to the use of anoligomer (compound) as defined herein or a conjugate as defined hereinfor the preparation of a medicament for the treatment of a disease,disorder or condition as referred to herein.

The methods of the invention can be employed for treatment orprophylaxis against diseases caused by abnormal levels of Tau protein.In some embodiments, diseases caused by abnormal levels of Tau proteinare tauopathies. In certain embodiments, tauopathies include Alzheimer'sdisease, progressive supranuclear palsy, dementia pugilistica (chronictraumatic encephalopathy), frontal temporal dementia, parkinsonismlinked to chromosome 17, Lytico-Bodig disease (Parkinson-dementiacomplex of Guam), Tangle-predominant dementia, ganglioglioma,gangliocytoma, meningioangiomatosis, subacute sclerosingpanencephalitis, lead encephalopathy, tuberous sclerosis,Hallervorden-Spatz disease, Pick's disease, corticobasal ganglionicdegeneration, argyrophilic grain disease, corticobasal degeneration,lipofuscinosis, frontotemporal dementia, supranuclear palsy, downsyndrome, and frontotemporal lobar degeneration.

In certain embodiments, the disease or condition for treatment orprophylaxis is a neurological disorder. In other embodiments, theneurological disorder is selected from progressive supranuclear palsy,frontotemporal dementia-tau (FTD-tau), frontotemporal dementia andparkinsonism linked to chromosome 17 (FTDP-17), corticobasaldegeneration (CBD), traumatic brain injury, chronic traumaticencephalopathy, HIV associated neurocognitive disorders, Argyrophilicgrain disease, Down syndrome-Alzheimer's disease, Amnestic mildcognitive impairment-Alzheimer's disease, Parkinson's disease dementia,Hallervorden-Spatz disease (Pantothenate kinase-associatedneurodegeneration), Niemann Pick disease type C, Myotonic dystrophy,Amyotrophic lateral sclerosis, Parkinson's disease, Huntington'sdisease, Hemimegalencephaly, Tuberous sclerosis complex, Focal corticaldysplasia type 2b, or Ganglion cell tumors. In certain embodiments, thedisease or condition is an epileptic disorder without tauopathy, e.g.,Dravet Syndrome (severe myoclonic epilepsy of infancy), Temporal lobeepilepsy, Ohtahara syndrome (early infantile epileptic encephalopathywith suppression bursts), Lafora body disease, Generalized epilepsy withfebrile seizures, Infantile spasms (West syndrome), Lennox Gastautsyndrome, Angelman Syndrome, Rett Syndrome, Landau Kleffner syndrome,focal seizures, simple focal seizures (no loss of consciousness), focaldyscognitive seizures (impairment of consciousness), focal seizureevolving to generalized tonic-clonic (GTC) convulsions, generalizedseizures (convulsive or non-convulsive with bilateral dischargesinvolving subcortical structures), absence seizures, myoclonic seizures,clonic seizures, tonic seizures, tonic-clonic seizures, atonic seizures,an autistic disorder, an autism spectrum disorder (e.g., as defined inthe Diagnostic and Statistical Manual of Mental Disorders V (DSM-V)), anAsperger's disorder, a pervasive developmental disorder, or anycombination thereof.

In certain embodiments, the neurological disorder is a neurodegenerativedisorder, an epileptic disorder, an idiopathic adult epileptic disorder,or any combination thereof. In other embodiments, the disease orcondition is a neurodegenerative disorder with tauopathy (i.e., aneurodegenerative disease which involves accumulation of tau protein inthe brain), an epileptic disorder with tauopathy (an epileptic disorderwhich involves accumulation of tau protein in the brain), an epilepticdisorder without tauopathy (an epileptic disorder which does not involveaccumulation of tau protein in the brain), an idiopathic adult epilepticdisorder without tauopathy (an idiopathic adult epileptic disorder whichdoes not involve accumulation of tau protein in the brain), or anycombination thereof. In certain other embodiments, the disease orcondition for treatment or prophylaxis is a neurodegenerative diseasewith tauopathy, e.g., progressive supranuclear palsy, frontotemporaldementia-tau (FTD-tau), frontotemporal dementia and parkinsonism linkedto chromosome 17 (FTDP-17), corticobasal degeneration (CBD), traumaticbrain injury, chronic traumatic encephalopathy, HIV associatedneurocognitive disorders, Argyrophilic grain disease, Downsyndrome-Alzheimer's disease, Amnestic mild cognitiveimpairment-Alzheimer's disease, Parkinson's disease dementia,Hallervorden-Spatz disease (Pantothenate kinase-associatedneurodegeneration), Niemann Pick disease type C, Myotonic dystrophy,Amyotrophic lateral sclerosis, Parkinson's disease or Huntington'sdisease. In certain embodiments, the disease or condition for treatmentor prophylaxis is an epileptic disorder with tauopathy, e.g.,Hemimegalencephaly, Tuberous sclerosis complex, Focal cortical dysplasiatype 2b, or Ganglion cell tumors. In certain embodiments, the disease orcondition is an epileptic disorder without tauopathy, e.g., DravetSyndrome (severe myoclonic epilepsy of infancy), Temporal lobe epilepsy,Ohtahara syndrome (early infantile epileptic encephalopathy withsuppression bursts), Lafora body disease, Generalized epilepsy withfebrile seizures, Infantile spasms (West syndrome), Lennox Gastautsyndrome, Angelman Syndrome, Rett Syndrome, or Landau Kleffner syndrome.In certain embodiments, the disease or condition for treatment orprophylaxis is an idiopathic adult epileptic disorder without tauopathy,e.g., focal seizures, simple focal seizures (no loss of consciousness),focal dyscognitive seizures (impairment of consciousness), focal seizureevolving to generalized tonic-clonic (GTC) convulsions, generalizedseizures (convulsive or non-convulsive with bilateral dischargesinvolving subcortical structures), absence seizures, myoclonic seizures,clonic seizures, tonic seizures, tonic-clonic seizures or atonicseizures. In certain embodiments, the neurological disorder fortreatment or prophylaxis is an autistic disorder, an autism spectrumdisorder (e.g., as defined in the Diagnostic and Statistical Manual ofMental Disorders V (DSM-V)), an Asperger's disorder or a pervasivedevelopmental disorder.

Alternatively stated, in some embodiments, the invention is furthermoredirected to a method for treating abnormal levels of Tau protein, themethod comprising administering a oligomer of the invention, or aconjugate of the invention or a pharmaceutical composition of theinvention to a patient in need thereof.

The invention also relates to an oligomer, a composition or a conjugateas defined herein for use as a medicament.

The invention further relates to use of a compound, composition, or aconjugate as defined herein for the manufacture of a medicament for thetreatment of abnormal levels of Tau protein or expression of mutantforms of Tau protein (such as allelic variants, such as those associatedwith one of the diseases referred to herein).

A patient who is in need of treatment is a patient suffering from orlikely to suffer from the disease or disorder.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986);); Crooks, Antisense drug Technology:Principles, strategies and applications, 2^(nd) Ed. CRC Press (2007) andin Ausubel et al. (1989) Current Protocols in Molecular Biology (JohnWiley and Sons, Baltimore, Md.).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Construction of Oligomers

A number of oligomers were designed to target the 3′ UTR of MAPTpre-mRNA. See FIG. 1 for genomic MAPT sequence. For example, theoligomers were constructed to target nucleotides 134,821-138,940 of SEQID NO: 1. The exemplary sequences of the oligomers are described inFIGS. 2, 3, 6, and 7. In some embodiments, the oligomers were designedto be gapmers or mixmers. FIG. 2 shows non-limiting examples of theoligomer design for selected sequences. The same methods can be appliedto any other sequences disclosed herein. The gapmers were constructed tocontain locked nucleic acids—LNAs (upper case letters). For example, agapmer can have Beta-deoxy LNA at the 5′ end and the 3′ end and have aphosphorothioate backbone. But the LNAs can also be substituted with anyother nucleotide analog and the backbone can be other type of backbone(e.g., a phosphodiester linkage, a phosphotriester linkage, amethylphosphonate linkage, a phosphoramidate linkage, or combinationsthereof).

The oligomers were synthesized using methods well known in the art.Exemplary methods of preparing such oligomers are described inBarciszewski et al., Chapter 10—“Locked Nucleic Acid Aptamers” inNucleic Acid and Peptide Aptamers: Methods and Protocols, vol. 535,Gunter Mayer (ed.) (2009), the entire contents of which is herebyexpressly incorporated by reference herein.

In FIG. 2, in the Sequence designation, upper case designates a modifiednucleotide such as an LNA nucleotide (either Beta-D-Oxy, Alpha-L-Oxy,Beta-D-Amino or Beta-D-Thio LNA or other modified nucleotide such ascEt, cMOE, UNA or ENA) and lower case designates a DNA nucleotide. Thusa sequence represented by TAGccctaaagtcCCA (SEQ ID NO: 53, i.e.,ASO-000389) represents a 3-10-3 16 mer modified nucleotide-DNA-modifiednucleotide gapmer with a 5′-T and 3′-A, such as a 3-10-3 LNA-DNA-LNAgapmer. Some oligomers can be an alternating flank gapmer as describedelsewhere herein. In some embodiments, selected examples of alternatingflank gapmers having a 7 nucleotide gap are ASO-002399, ASO-002482,ASO-002437, and ASO-002425. Any one of the oligomer sequences disclosedherein can have the alternating flank gapmer design shown in thefigures. In addition, any one of the oligomer sequences disclosed hereincan have the chemical structure shown in FIGS. 2, 16B, and 20B.

In FIG. 2, the following designate the components of theoligonucleotides of the present invention, with oligonucleotides alwaysdepicted in the 5′ to 3′ direction. Therefore, the 5′ end of an oligomerhybridizes to the pre-mRNA end number in the table and the 3′ end of theoligomer hybridizes to the pre-mRNA start number in the figure. Areference to a SEQ ID number includes a particular sequence, but doesnot include an oligomer design or its chemical structure.

Beta-D-oxy LNA nucleotides are designated by OxyB where B designates anucleotide base such as thymine (T), uridine (U), cytosine (C),methylcytosine (MC), adenine (A) or guanine (G), and thus include OxyA,OxyT, OxyMC, OxyC and OxyG.

Alpha-L-oxy LNA nucleotides are designated by AlfaOxyB where Bdesignates a nucleotide base such as thymine (T), uridine (U), cytosine(C), methylcytosine (MC), adenine (A) or guanine (G), and thus includeAlfaOxyA, AlfaOxyT, AlfaOxyMC, AlfaOxyC and AlfaOxyG. The letter M or mbefore C or c indicates 5-methylcytosine.

Beta-D-Amino LNA nucleotides are designated by AminoB where B designatesa nucleotide base such as thymine (T), uridine (U), cytosine (C),methylcytosine (MC), adenine (A) or guanine (G), and thus includeAminoA, AminoT, AminoMC, AminoC and AminoG. The letter M or m before Cor c indicates 5-methylcytosine. Some examples of the oligomersincluding 5 methylcytosine include ASO-002672, ASO-002658, ASO-002622,ASO-002629, ASO-002621, ASO-002665, and ASO-002630. See FIG. 2.

Beta-D-Thio-LNA nucleotides are designated by ThioB where B designates anucleotide base such as thymine (T), uridine (U), cytosine (C),methylcytosine (MC), adenine (A) or guanine (G), and thus include ThioA,ThioT, ThioMC, ThioC and ThioG. The letter M or m before C or cindicates 5-methylcytosine.

DNA nucleotides are designated by DNAb, where the lower case bdesignates a nucleotide base such as thymine (T), uridine (U), cytosine(C), 5-methylcytosine (MC), adenine (A) or guanine (G), and thus includeDNAa, DNAt, DNAc, DNAmc and DNAg. The letter M or m before C or cindicates 5-methylcytosine.

The letter “s” after the nucleotide designation indicatesphosphorothioate linkage whereas absence of “s” indicates phosphodiesterlinkage.

Thus a 3-10-3 beta-D-oxy LNA-DNA-beta-D-oxy LNA gapmer with sequenceATTtccaaattcaCTT, with full phosphorothioate internucleotide linkageswould be designated OxyAs OxyTs OxyTs DNAts DNAcs DNAcs DNAas DNAasDNAas DNAts DNAts DNAcs DNAas OxyMCs OxyTs OxyT. In some embodiments,the oligomers have a mix of phosphorothioate and phosphodiesterinternucleotide linkages. Examples of the oligomers having a mix ofphosphorothioate and phosphodiester internucleotide linkages include,but are not limited to, ASO-002625, ASO-002675, ASO-002633, ASO-002640,ASO-002632, ASO-002647, ASO-002655, ASO-002641, ASO-002648, ASO-002666,ASO-002659, ASO-002652, ASO-002645, ASO-002638, ASO-003270, ASO-003269,ASO-003268, ASO-002673, ASO-002661, ASO-002654, ASO-002668, ASO-002676,AS-002669 and ASO-002662. See FIG. 2.

Preparation of Oligos with Mismatches

Oligos having mismatched bases at different locations were also preparedusing standard methods well known in the art. Examples of oligomers withmismatched bases are provided in FIG. 2 or 3 as “mm.” The specificmismatched basepair are bolded, underlined, italicized, and highlighted.

Example 2 In Vitro Reduction in Tau Protein

Each of the oligomers targeting the 3′ UTR of an MAPT transcript wastested for its ability to decrease Tau protein in mouse primary neuronsexpressing the entire human MAPT gene as a bacmid containing transgene(C57-b16 BAC-Tg hTau; Polydoro et. al., J. Neurosci. (2009) 29 (34):10747-9). Primary hTau mouse embryonic forebrain neuronal cultures donot express endogenous mouse tau as mouse tau was knocked out. Primaryneurons were generated by papain digestion according to manufacturer'sprotocol (Worthington Biochemical Corporation, LK0031050). Briefly,forebrains were dissected from hTau mouse E18 BAC-Tg embryos expressingthe entire human microtubule-associated protein Tau (MAPT) gene on amurine MAPT-null background and were incubated at 37° C. for 30-45minutes in papain/DNase/Earle's balanced salt solution (EBSS) solution.After trituration and centrifugation of cell pellet, the reaction wasstopped by incubation with EBSS containing protease inhibitors, bovineserum albumin (BSA) and DNase. The cells were triturated and washed withNeurobasal (NB, Invitrogen) supplemented with 2% B-27, 100 μg/mlpenicillin, 85 μg/ml streptomycin, and 0.5 mM glutamine. The cells wereplated in supplemented NB media onto poly-D-lysine-coated 96-welloptical imaging plates (BD Biosciences) at 15,000 cells/well.

After obtaining the primary hTau mouse embryonic forebrain neuronalcultures expressing a human MAPT gene, the cultures were treated witholigomers to inhibit the Tau mRNA and protein expression. The cultureswere then subject to immunocytochemistry and imaging to measure theinhibition. One day post plating (DIV 1), half of the supplementedneurobasal (NB) media on the primary hTau mouse embryonic forebrainneuronal cultures was removed and replaced with supplemented NB mediacontaining various concentrations of LNA oligomers. Primary hTauneuronal cultures were cultured with LNA oligomers until 13 days postplating (DIV 13). On DIV 13, the cultures were rinsed with Dulbecco'sphosphate-buffered saline lacking calcium and magnesium (DPBS,Invitrogen) and fixed in 4% paraformaldehyde/4% sucrose/DPBS for 15 min.Cultures were rinsed and then blocked and permeabilized in DPBS plus0.1% Triton X-100 (TX-100) and 3% BSA for one hour at room temperature.Cultures were rinsed and then incubated for two hours at roomtemperature with primary antibody 1:500, Tau5 antibody to measure Tauprotein, Invitrogen AHB0042; and 1:500, β-III tubulin (TuJ-1) antibodyto measure neurite area, Abcam ab41489) in DPBS plus 3% BSA and 0.1%TX-100. Cultures were rinsed and incubated with Hoeschst 33342 nucleardye (1:800, Invitrogen) and AlexaFluor fluorescence-conjugated secondaryantibodies (Invitrogen, 1:500) in DPBS plus 3% BSA and 0.1% TX-100 forone hour at room temperature. Cultures were rinsed abundantly and storedin DPBS until imaging. Imaging was conducted using the Cellomics VTiautomated immunofluorescence imaging system. In brief, using untreatedwells, saturation levels for each fluorophore channel were set to 70%.Then 12 sequential images were acquired from each well, and totalfluorescence intensity and total fluorescence area were calculated forboth Tau and TuJ-1 proteins using the Cellomics VTi SpotDetector(version 4) image analysis software. To evaluate Tau protein reductionresulting from oligomer treatment, a Tau5 total fluorescenceintensity-to-Tuj-1 total fluorescence area ratio (Tau/TuJ-1) was createdfor each well and then all data were normalized to the average Tau/Tuj-1ratio of the untreated wells. TuJ-1 intensity acts as an internalstandard for each sample. To evaluate neurite/neuronal toxicity fromoligomer treatment, the Tuj-1 total fluorescence area from each well wasnormalized to the average Tuj-1 total fluorescence area of the untreatedwells. Nuclei counts from each well were also acquired as an alternativemeasure of toxicity associated with LNA oligomer treatment. Data areexpressed as mean±S.D. For immunocytochemistry, data points representthe mean±S.D. from wells treated in triplicate. Potency values weregenerated using wells treated with a broad concentration range of LNAoligomer, from which the resulting normalized Tau/Tuj-1 and Tuj-1 valueswere analyzed compared to normalized values from saline control samples.Analysis was done using non-linear regression with top and bottom valuesset at fixed values of 100% and 0%, respectively, where 100% inhibitionrepresents a complete reduction of signal compared to the control sample(FIG. 3). For qPCR, data were analyzed using a one-way ANOVA with aDunnett's multiple comparison test to compare saline- and LNAoligomer-treated groups. Statistical significance was set at a value ofp<0.05.

The reduction of Tau protein by each oligomer was compared with saline.The results of the Tau protein reduction compared to Saline are shown inFIG. 3. If the Tau protein level in antisense oligonucleotide treatedneurons was equal to or higher than in control cells, percent inhibitionis expressed as zero inhibition. The target regions to which antisenseoligomers are inhibitory are considered ‘hot-spots’ on the Tautranscript.

Oligomers were diluted in water and added to cells at 1 day post plating(DIV01) to a final concentration of 5 μM. For IC₅₀ determinations,neurons were treated with a top concentration of 5 μM and aconcentration response dilution of 1:3 was used to define the IC₅₀value. The calculated IC₅₀ value for certain oligomers is shown in FIG.6.

Example 3 Spontaneous Calcium Oscillation Measurement

The present application shows that a reduction of oscillations inintracellular free calcium concentration (calcium oscillation)corresponds to increased neurotoxicity of an oligomer to a cell. Theamount of reduction and how it corresponds to an increase inneurotoxicity can be determined as described herein. To measure primarycortical neuron spontaneous calcium oscillation, rat primary corticalneurons were prepared from Sprague-Dawley rat embryos (E19). Cells wereplated 25,000 cells/well onto 384 well poly-D-lysine coated fluorescentimaging plate reader (FLIPR plates) (Greiner Bio-One) in 25 μl/wellNeurobasal media containing B27 supplement and 2 mM glutamine. Cellswere grown for 11 days at 37° C. in 5% CO₂ and fed with 25 μl ofadditional media on DIV04 and DIV08 for use on DIV11. On the day of theexperiment, media was removed from the plate and the cells were washedonce with 50 μl/well of 37° C. assay buffer (Hank's Balanced SaltSolution with 2 mM CaCl₂ and 10 mM Hopes pH 7.4). Oscillations weretested in the presence and absence of 1 mM MgCl₂ (FIG. 4). Cells wereloaded with a cell permanent fluorescent calcium dye, fluo-4 AM (LifeTechnologies). Fluo-4 AM was prepared at 2.5 mm in DMSO containing 20%plutonic F-127 then diluted 1:1000 in assay buffer. Cells were incubated1 hr with 20 μl of 2.5 μM fluo-4 AM at 37° C. in 5% CO₂. After 1 hr 20μL of room temperature assay buffer was added and the cells were allowedto equilibrate to room temperature for 10 additional minutes and placedin the FLIPR. Baseline signal (measurement of intracellular calcium) wasread for 100 seconds (1 reading/second) before the addition ofanti-sense oligomers. Oligomers were added with a 384 well head in theFLIPR in 20 μl of assay buffer at 75 μM for a final concentration of 25μM. FLIPR signal was read for an additional 200 seconds (1reading/second) after the addition of oligomer. A second 5 minute postaddition plate read (300 one second points) on the FLIPR was conductedto allow for additional data capture. Raw data from the 5 minute readwas exported and, using Excel, spike amplitude and frequency wascalculated. Calculations were performed by measuring the average FLIPRsignal over the 300 second read for control (non-treated) wells. Fortreated wells, a scoring system was developed where a score of 1 wasgiven for each 1 second read where signal increase greater than 50% ofthe average control value (calculated above). A score of 0 was given foreach 1 second read that increased less than 50% of average controlvalue. For each treatment a total score was calculated and converted topercent control for graphical purposes. If the antisense oligomerproduced a calcium oscillation response greater than that of AMPA alone,percent of control is expressed as greater than 100% (FIG. 6).

Effect of oligomers on primary neuronal spontaneous calcium oscillationswas measured under two conditions, in the presence and absence of 1 mMMgCl₂ as a source of Mg²⁺ ions, as described previously (Murphy et.al.,J. Neurosci. 12, 4834-4845 (1992)). This was done to isolatedN-methyl-D-aspartate (NMDA)- andα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptormediated calcium oscillations. Data presented in FIG. 4 show that,addition of the AMPA receptor antagonist6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 3 μM) reduced calciumoscillations by 20%, representing the total AMPA response in the assay(FIG. 4 AMPA labeled bar shown). Calcium oscillations were reducedfurther, by about 80%, when (NMDA) receptor function was blocked by 1 mMMgCl₂ (FIG. 4 NMDA labeled bar shown).

Antisense oligomer inhibition of spontaneous calcium oscillationsmediated by either NMDA or AMPA was assessed in the presence or absenceof 1 mM MgCl₂ (representing 100% control in each case; FIG. 5). Additionof 25 μM antisense oligomers (ASO) inhibited AMPA receptor but not NMDAreceptor mediated oscillations (FIG. 5). ASO, and other oligos thatbehaved similarly, were shown to negatively impact central nervoussystem (CNS) network activity in vivo and electrophysiologic spontaneousneuronal activity in vitro (data not shown). The impact of Tau antisenseoligonucleotides on spontaneous calcium oscillations in primary neuronsis summarized in FIG. 6. See Murphy et al., J. Neurosci. 12, 4834-4845(1992).

Calcium oscillation reduction was measured for the oligomers of theinvention and summarized in FIG. 6. The oligomers showing greater than25% of control in the calcium oscillation assay were selected forfurther analysis.

Example 4 Sequence Score Calculation

The present application also shows that the sequence score of anoligomer, as calculated herein, corresponds to the neurotoxicity of theoligomer. In certain aspects of the invention, the higher the sequencescore the less neurotoxic the oligomer. Different cut off values, overwhich the sequence score indicates that the oligomer has reducedneurotoxicity, can be determined as described herein.

The sequence score of each oligomer was calculated to predict thesuitability and neurotoxicity of the oligomers. Sequence score is amathematical calculation determined for all oligomers and is based onthe percent of G and C nucleotides, or analogs thereof, within a givenoligomer sequence. The following formula was applied to all oligomers inorder to calculate sequence score:

$\begin{matrix}\frac{{{number}\mspace{14mu}{of}\mspace{14mu} C\mspace{14mu}{nucleotides}} - {{number}\mspace{14mu}{of}\mspace{14mu} G\mspace{14mu}{nucleotides}}}{{nucleotide}\mspace{14mu}{length}} & (I)\end{matrix}$

An example calculation is given for oligomer ASO-000013 (SEQ ID NO: 686;sequence score 0.25): ATTtccaaattcaCTT: 4-0/16=sequence score of 0.25.

The sequence score of the selected oligomers were calculated for furtherstudies.

To determine the cut off value for the sequence score, an in vivotolerability study was performed as shown in Example 5.

Example 5 In Vivo Tolerability and In Vivo Tau mRNA Reduction

The in vivo tolerability of the oligomers was tested to see how theoligomer was tolerated when injected into an animal.

Subjects

In vivo tolerability of the oligomers were tested in mice and rats.Animals for Tau qPCR and behavioral studies were adult, C57B1/6J femalemice (20-30 g; Jackson Laboratories, Bar Harbor, Me.) housed 3-4 percage. Animals were held in colony rooms maintained at constanttemperature (21±2° C.) and humidity (50±10%) and illuminated for 12hours per day (lights on at 0600 hours). In some cases, male and femaletransgenic mice (30-40 g) expressing a tau transgene derived from ahuman PAC, H1 haplotype driven by the tau promoter (Polydoro et. al., J.Neurosci. (2009) 29(34): 10741-9), and in which the native mouse Taugene was deleted, were used to assess pharmacodynamic endpoints andtissue drug concentrations. For intrathecal infusion studies, femaleSprague-Dawley rats (180-225 g at testing; Harlan) were singly housed incolony rooms maintained at a constant temperature (21±2° C.) andhumidity (50±10%) and illuminated for 12 hours per day (lights on at0600 h). All animals had ad libitum access to food and water throughoutthe studies. Behavioral studies were conducted between 0700 and 1500hours. Animals were maintained in accordance with the guidelines of theAnimal Care and Use Committee of the Bristol-Myers Squibb Company, andthe “Guide for Care and Use of Laboratory Animals” published by theNational Institutes of Health. Research protocols were approved by theBristol-Myers Squibb Company Animal Care and Use Committee.

Administration Routes—Intra-Cerebroventricular or IntrathecalInjections.

The oligomers were administered to mice by eitherintracerebroventricular (ICV) injection or intrathecal injection.Intracerebroventricular injections were performed using a Hamilton microsyringe fitted with a 27 or 30-gauge needle, according to the method ofHaley and McCormick. The needle was equipped with a polyethylene guardat 2.5 mm from the tip in order to limit its penetration into the brain.Mice were anesthetized using isoflurane anesthetic (1.5-4%). The mouseto be injected, weighing 20-30 g, was held by the loose skin at the backof the neck with the thumb and first fingers of one hand. Applyinggentle but firm pressure, the head of the animal was then immobilized bypressing against a firm flat level surface. The needle tip was theninserted through the scalp and the skull, about 1 mm lateral and 1 mmcaudal to bregma. Once the needle was positioned, antisenseoligonucleotide was given in a volume of 5 microliters in saline vehicleand injected into the right (or left) lateral ventricle over 20-30seconds. The needle was left in place for 10 seconds before removal.This procedure required no surgery or incision. Animals were warmed onheating pads until they recovered from the procedure. Brain tissue(right, frontal cortical region) was collected on dry ice or RNAlaterfor drug concentration analysis and Tau qPCR respectively at multipletime points following dosing, e.g., 1 week through 16 weeks post-dosing.

For intrathecal (IT) injections of mice, animals were maintained underlight isoflurane anesthesia (1.5-5%). The mouse was held securely in onehand by the pelvic girdle and inserting a 30 G ½ inch needle connectedto a Hamilton syringe into the tissue between the dorsal aspects of L5and L6, perpendicular to the vertebral column. When the needle entersthe subarachnoid space, a sudden lateral movement of the tail wasobserved. This reflex was used as an indicator of successful placementof the needle for IT administration. A 5-10 μL volume of antisenseoligonucleotide was injected slowly (over approximately 60 seconds) intothe subarachnoid space.

For intrathecal injections in rat, intrathecal catheters were surgicallyimplanted using methods described by Yaksh and Rudy, Physiol. Behay.(1976) 17(6): 1031-6. The rat was mounted to a stereotaxic frame withisoflurane anesthesia maintained through a nose cone. A skin incisionwas made beginning approximately at the line joining the ears andextending caudally about 3 cm along the midline. The muscle where itattached to the occipital crest of the skull was cut about 3 mm lateralon both sides of the muscle midline. Using retractors or forceps, themuscle was peeled caudally to expose the cisternal membrane at the baseof the skull. The fascia and tissue were carefully removed from themembrane. The bent beveled end of a 16-22 gauge needle was used to makea 1-2 mm lateral incision in the cisternal membrane. A sterilized ITcatheter, made of polyethylene tubing (PE10 tubing stretched toapproximately 1.3 mm outer diameter), was inserted through the incisionand carefully advanced caudally through the subarachnoid space while itwas rotated between thumb and forefinger and while the base of the tailwas gently pulled to align the spinal cord using the other hand. If anyresistance was encountered, the catheter was retracted slightly, andslowly advanced again. Once the catheter had been advanced to thedesired area, it was flushed with 20 μL sterile saline and the cranialend was passed through the skin using a 19 gauge needle about 1 cm fromthe incision. The catheter was plugged with a pin. Rats were given oralantibiotics for 5 days following the surgery. At least five days aftersurgery, a single antisense oligonucleotide injection was diluted inwater and delivered via a programmable infusion pump (Knopf) at a rateof 10 μl/minute in a volume of 10 to 50 μl. A brief saline flush of 5 ulwas given just prior to the antisense oligonucleotide delivery and a 10μl saline flush was given just following the oligonucleotide delivery ata rate of 10 μl minute to cover the dead volume of the catheter (6-7μl). A saline flush of 20 ul was also given to animals 1-2×/week untilused for an experiment.

Acute Tolerability Behavioral Assessments

For one hour following the single injection of antisense oligonucleotideICV (intra-cerebroventricular) or IT (intrathecal), animals wereobserved for behavioral side effects and scored for the severity of sideeffects on a scale of zero (no side effects) to 20 (convulsionsresulting in euthanasia). The tolerability scale was divided into 5neurobehavioral categories: 1) hyperactivity 2) decreased activity andarousal 3) motor dysfunction/ataxia 4) abnormal posture and breathingand 5) tremor/convulsions. Each category was scored on a scale of 0-4,with the worst possible total score of 20. Animals were observed forchanges in behavior in the home cage, and then they were removed fromthe home cage for more detailed observations which included measurementof grip strength and righting reflex.

Novel Object Recognition

Short term recognition memory was measured using the novel objectrecognition (NOR) task. NOR testing was based on the spontaneousbehavior of rodents to explore a novel object more than a familiar one(Dodart et. al., Neuroreport (1997) 8(5): 1173-8; Ennaceur and Delacour,Behav. Brain Res. (1988) 31 (1):47-59). After a one hour retentioninterval between training (T1) and testing (T2) sessions, miceremembering the objects from the training session will show a preferencefor the novel object on the test session. For these experiments, animalswere handled for 3 days and habituated to the chamber (48 cm×38 cm×20cm) on the day prior to the test session. The chamber was made ofpolyethylene and lined with vinyl flooring. On the test day, animalswere placed in the rectangular test chamber and allowed to explore twoidentical objects (7.6 cm high×5.1 cm wide) for a 15 minute trainingperiod. One hour later, mice were placed back into the test chamber fora 10 minute test session, this time with one object they had observedduring training and one novel object. Objects were cleaned thoroughlywith 25% ethanol between training and testing sessions and betweensubjects, and were cleaned again at the end of the day with milddetergent. Object exploration was only considered when the animal's nosewas pointed at the object. Exploration was recorded using ObjectScantracking software (Cleversys, Reston, Va.). Data are reported as percentof time spent exploring objects (i.e., novel time/novel+familiar time*100).

Morris Water Maze

Spatial learning and memory was assessed based on Morris Water Mazeassay (Morris J. Neurosci. (1984) 11(1):47-60). Water maze represents apool with the diameter of 120 cm. Water was made opaque using white,non-toxic tempura paint (20° C.±1). The pool was surrounded withdistinct extra-maze cues.

Prior to hidden platform training, all mice were exposed to the watermaze pool by allowing them to swim down the rectangular channel during 2pre-training trials. The escape platform was placed in the middle of thechannel. If a mouse was not able to find and mount the platform during60 sec trial, it was guided to it and allowed to sit for up to 10 sec.After pre-training, mice underwent hidden platform training, duringwhich a 10×10 cm platform was submerged 1.5 cm below the surface. Theplatform location remained the same throughout training whereas the droplocation varied randomly between the four daily trials as well as acrossthe 4 days of training. Mice received 2 sessions per day for 4consecutive days. Each session consisted of 2 trials with a 10-mininter-trial interval. The maximum time allowed per trial was 60 sec. Ifa mouse did not find or mount the platform, it was guided to theplatform by the experimenter. All mice were allowed to sit on theplatform for 10 sec after each training trial.

For probe trials, the platform was removed and each mouse was allowed toswim for 60 sec. The drop location for the probe trials was 180° fromthe platform location used during hidden platform training. After 60sec, mice were guided to the platform location before retrieval from thepool. For early memory retrieval mice were probed 2 h after the lasthidden platform training; long term memory recall was assessed 16 hfollowing the last hidden platform training. 2 h following the 16 hprobe trial, all mice underwent the visible platform training, where alocal cue (pole built using legos) was placed above the hidden platform.Mice were given 2 training trials. All behavior was recorded with avideo tracking system (Cleversys, Inc). Escape latencies, distancetraveled, swim paths, swim speeds, and platform crossings were recordedautomatically for subsequent analysis.

Catwalk

The Catwalk (Noldus, The Netherlands) is an automated and computerizedgait-analysis technique that allows objective quantification of multiplestatic and dynamic gait parameters. Mice were placed on one end of thecatwalk and allowed free exploration for 3 min or until they have 5compliant trials, whichever comes first. Data were exported andclassified using the Catwalk software. An average of classified trialswas used for data analysis. Measures of interest include but are notlimited to: print position or the distance between the position of thehind paw and previous placement of the ipsilateral front paw, initialand terminal dual stances, paw swing speed, and paw stand or theduration of paw contact with the glass plate in a step cycle.

Behavioral Statistics

Statistical analyses for all behavioral tests were conducted usingGraphPad Prism (GraphPad Software, Inc., La Jolla, Calif.). For NOR,data were analyzed using either a paired t-test for within-groupanalyses or by an ANOVA followed by a Dunnett's post-hoc test forbetween group analyses. For MWM, a repeated MWM ANOVA was used toanalyze the acquisition phase and a one-way ANOVA followed by Dunnett'spost-hoc for probe trial analyses.

Brain Tau mRNA Analysis

Brain Homogenization

Mouse brain tissue was homogenized in a 10× volume of a highsalt/sucrose buffer (10 mM Tris-HCl, pH 7.4, 800 mM NaCl, 10% sucrose(w/v), 1 mM EGTA) supplemented with phosphatase inhibitor cocktail sets2 and 3, 1 mM PMSF (Sigma, Saint Louis, Mo.), and complete proteaseinhibitor cocktail EDTA-free (Roche, Indianapolis, Ind.) using a QuiagenTissueLyzer II. The homogenate was centrifuged at 20,000×g for 20minutes at 4° C. The supernatant was centrifuged at 100,000×g for 1 hourat 4° C. and the supernatant was analyzed.

RT-PCR Assays

For cDNA synthesis and subsequent PCR, 300 ng of RNA from brain tissuewas added to 1 well of a 96 well plate (Axygen, PCR-96-C-S). To eachwell 7.5 μl of master mix (54, of 2.5 mM NTP mix and 2.54, randomprimers per reaction) was added and the plate was centrifuged at 1000rpm and placed in thermocycler for 3 min at 70° C. Plates wereimmediately cooled on ice and 4 μl of reaction master mix was added.Prior to PCR, plates were briefly centrifuged to collect sample inbottom of well. cDNA synthesis was carried out at 42° C. for 60 min, 95°C. for 10 min followed by a hold at 4° C. cDNA Samples were diluted 1:3with molecular biology grade water and stored at −20° C. until furtheruse.

For PCR, each sample was run in triplicate with two probe sets (MAPT:Taqman Expression assays Hs00902193_ml; RhoA: Taqman Expression assays;GAPDH Taqman Expression assays Hs01922876_ul). To each reaction 4 μl ofpreviously diluted cDNA and 6 μL of master mix was added and plates werecentrifuged. Samples were incubated at 95° C. for 20 sec follow by 40cycles at 95° C. for 1 sec and 60° C. for 20 sec.

Data was analyzed using the delta delta Ct method where each sample isfirst normalized to GAPDH and then expressed as percent of untreatedcontrol animals (see FIG. 7).

Results

In vivo cumulative tolerability threshold following an ICV injection of100 m of an antisense oligonucleotide was set at 4. The correlationanalysis in FIG. 8A shows that the oligomers having in vivo tolerabilitylower than 4 tend to have a sequence score equal to or higher than 0.2.Squares in FIG. 8B represent oligomers prioritized based on not only onin vitro Tau protein reduction, but also on primary neuronal health andactivity assessed by tubulin and spontaneous calcium oscillationscriteria outlined above. In vitro potency data concorded well with invivo tau mRNA reduction allowing for additional prioritization ofoligomers. Potent LNA oligonucleotides targeting MAPT at or partiallyoverlapping nucleotides in the 3′UTR were identified and found to bewell tolerated in primary neurons in vitro and following ICVadministration in vivo (See FIG. 7).

The in vivo acute tolerability score and brain tau mRNA % control datashown in FIG. 7 show that selected oligomers that hybridize to targetMAPT mRNA sequences are both well tolerated and potently reduce Tau mRNAin vivo (e.g., ASO-000013-ATTtccaaattcaCTT-SEQ. ID No. 686:138,888-138,903).

Example 6 Oligomer Prioritization

The assays described herein can be used in combination to selectedoligomers for further testing. Properties of selected oligomers can bedescribed as shown in Table 1. Based on these criteria, certainoligomers were selected for additional dose-response testing in vitroand in vivo.

TABLE 1 Summary of criteria used to prioritize oligomers for additionaltesting. Assay Prioritization Criteria Tau protein reduction >70%reduction in Tau protein (5 μM oligomer) Calcium oscillations <25%reduction in calcium oscillations Sequence score Sequence score ≥0.20

In another embodiment, oligomers can be selected based on the followingcharacteristics: (1) Tau protein reduction >30% reduction in Tau protein(5 μM oligomer); (2) calcium oscillations <25% reduction in calciumoscillations; and (3) sequence score equal to or higher than 0.2.

Example 7 In Vivo Data

Oligomers were injected into animals to determine their effect on Tauexpression and on the behavioral properties of the animal.

Research Animals and Administration Routes

The animals used in this Example are the same mice and rats described in

Example 5 and were handled in the same manner as described in Example 5.Animals were injected as described in Example 5.

RT-PCR assays were performed as described in Example 5.

Running Wheel Assay

The Home Cage Running Wheel assay measures spontaneous activity in avoluntary free-spinning running wheel (Columbus Instruments). Each wheelhas a magnetic sensor that connects to a computer interface and recordswheel revolutions at user-specified intervals. In this study, mice wereplaced individually into cages with a running wheel and wheel rotationswere monitored continuously in 15 min increments. To allow forhabituation and establish baseline activity levels, control and testmice were tested over 7 days, after which they were transferred intoclean cages and dosed with either saline or 100 ug ASO-000774 by ICVinjection. Two weeks post treatment mice were returned to the runningwheel cages to evaluate treatment effects over 7 days.

Brain Tau mRNA Analysis

Brain Homogenization

Mouse brain tissue was homogenized as described in Example 5. Survivaland Febrile Seizure Data (Gheyara et. al., Ann Neurol. 2014; 76(3):443-456.)

Heat-Induced Seizures: Seizures were induced in P30-45 mice using a heatlamp as described (Oakley et al., Proc Natl Acad Sci USA 2009;106:3994-3999) except that a 2-minute acclimation period was used.Survival and febrile seizure data were analyzed by Cox proportionalhazards regression using the R survival package (Therneau T M. Survivalanalysis. R package version 2.37-4 ed2013) and corrected for multiplecomparisons with the method of Holm (Holm S. Scand J Stat 1979; 6:65-70)For analysis of drug-induced epileptic activity in brain slices, alinear mixed effects model (Laird et al., Biometrics 1982; 38:963-974.)was fitted using the R package. Random intercepts were included for eachmouse and each genotype such that multiple comparison corrections werenot needed due to “partial pooling.” Five thousand draws were obtainedof parameter estimates, and 95% confidence intervals (CIs) wereestimated as the 2.5th and 97.5th quantiles of these draws. Probabilityvalues were calculated by inverting the simulated CIs around thedifferences. Analyses of log(spike frequency 10.1) and log(burstfrequency 10.1) were conducted separately.

CSF Collection

All animal protocols were approved by the Wallingford BMS Animal Careand Use Committee. CSF was collected from the cisterna magna of micefollowing exsanguination as described by Barten, et al., J Alz. Res. 24:127-141 (2011). In brief, CSF was collected with a P20 pipettor afterpuncturing the dura with a 30 gauge needle under a dissectingmicroscope. Body temperature was monitored and maintained at normallevels using heating pads and lamps. CSF was collected from rats afterexposure of the cisterna magna and withdrawal using a 1 ml insulinsyringe. CSF was placed on ice, centrifuged briefly to remove any redblood cells, transferred to another tube while measuring the volume, andfrozen on dry ice. CSF Tau protein reduction measured by Tau ProteinEnzyme-Linked ImmunoSorbant Assay (ELISA described below) was observedafter 4 weeks following a single bolus ICV injection of ASO-000013 (datanot shown).

Tau Protein Enzyme-Linked ImmunoSorbant Assay (ELISA)

For brain tissue, BT2 (antibody to Tau amino acid 194-198, ThermoScientific) was used to coat 96 well black ELISA plates (Costar) at aconcentration of 2.5 μg/ml for 1 hour at 37° C. After washing in TBST,the plates were blocked with 3% bovine serum albumin in TBS. Recombinanthuman Tau441 (rPeptide; Bogart, Ga.) or a 1:5000 dilution of the brainhomogenates were diluted in 1% BSA+0.05% Tween-20 in TBS. Alkalinephosphatase conjugated Tau-5 (antibody to Tau amino acid 210-230,Covance, Emeryville, Calif.) was added to the samples at a 1:2000dilution for co-incubation overnight at 4° C. with shaking. Afterwashing in TBST, the signal was amplified with the Tropix CDP Stardetection reagent from Applied Biosystems. The chemiluminescent signalwas read on an Envision (Perkin Elmer). For CSF samples, this ELISA wasdone in a 384 well format to minimize the volume of CSF needed. 10 μl ofa 1:2 dilution of CSF was added to each well.

In-Situ Hybridization

In-situ hybridization (ISH) detection of Tau mRNA or ASO-000013 wasperformed on 20 μm fresh frozen brain sections mounted. Slides werethawed, fixed in 4% paraformaldehyde for 10 minutes at room temperature,washed in phosphate buffered saline (PBS) and acetylated with 0.25%acetic anhydride/0.1M triethanolamine for 10 minutes at room temperature(RT). Following PBS washes, each slide was pre-hybridized in 0.7 mlpre-warmed hybridization buffer (HB), 50% formamide/5× saline sodiumcitrate (SSC), 100 μg/ml yeast tRNA, 1×Denhardt's, for 30 minutes at 67°C. 5′ FAM-labeled ASO-000013 sense probe (complementary all LNA probe,ASO-000067=SPC-11404) was heated to 90° C. for 4 minutes, cooled on icethen diluted in HB. Slides were hybridized in 0.45 ml for 30 minutes at67° C. with a hybrislip (Electron Microscopy Sciences, Hatfield, Pa.).They were subsequently dipped in 0.1×SSC then washed three times in0.1×SSC at 67° C. Slides were then treated in 3% hydrogen peroxide for10 minutes, washed in PBS, and blocked for 15 minutes at RT in 0.1MTris-HCl, pH 7.5, 0.15M NaCl, 0.5% blocking agent (FP1020, Perkin ElmerWaltham, Mass.). This was followed by incubation in rabbitanti-fluorescein-horse radish peroxidase for 30 minutes at RT. FollowingTBST washes (Tris buffered saline with 0.05% Tween 20), tyramide signalamplification was performed (TSA Plus, Perkin Elmer). Slides were washedin TBST, nuclei stained using DAPI, and coverslip mounted using ProlongGold (Invitrogen, Carlsbad, Calif.). For chromogenic detection, slides(post-TSA washes) were incubated for a second time withanti-fluorescein-HRP for 30 minutes at RT, washed in TBST and developedusing DAB substrate (Quanto, Thermo Scientific, Freemont, Calif.). TaumRNA and ASO-00013 oligomer ISH indicate uniform distribution of taumRNA reduction and oligomer across the mouse brain following a singleICV bolus injection of 100 μg ASO-000013 (data not shown).

Results

In vivo reduction of human tau mRNA level was measured in mice after theadministration of various oligomers (FIG. 7). As shown in FIGS. 9A and9B, brain Tau mRNA and Tau protein reduction over time following asingle ICV bolus of 100 μg ASO-000013 (i.e., ATTtccaaattcaCTT, in whichthe upper case letters represent LNA nucleotides while the lower caseletters represent DNA nucleotides) administration into wild type C57mice (N=12). Tau mRNA expression (normalized to GAPDH) was measured at2, 4, 8 and 12 weeks post injection. Tau protein (% of saline) level wasmeasured at 2, 4, 8 and 12 weeks post injection. This oligomer produceda durable reduction in Tau mRNA and protein with Tau protein remainingreduced following 12 weeks post single bolus ICV injection. Otheroligomers defined within this invention exhibit more profound reductionsin Tau mRNA, as measured by qRT-PCR, and protein with durable tissueoligomer exposure (FIG. 10) as measured by ELISA (further describedbelow).

Tau mRNA Reduction

Oligonucleotides, or oligomers similar to ASO-000013, ASO-000757,ASO-000762, ASO-000761, ASO-000758, ASO-000760, and ASO-000759 showpotent knockdown of Tau protein in primary hTau neurons with goodtolerability in vitro and in vivo when administered directly into thecerebral spinal fluid (CSF) via intra-cerebroventricular or intrathecaldosing (see, e.g., FIG. 7. They also display robust, durable taureduction in the brain following intra-cerebroventricular administrationof 100 ug in C57 b16 mice (FIG. 7). Inhibition of calcium oscillationsin primary neurons was not observed in primary neurons treated withthese oligomers. This inhibition of calcium oscillations in primaryneurons was a strong indication of acute in vivo tolerability issuesrelated to network dysfunction when injected into CSF directly.

Oligomers like ASO-000013 produced sustained Tau reduction following a100 μg intra-cerebroventricular (ICV) bolus injection (see FIG. 9). 100μg/5 μl was injected into wt C57 mice, 3 Month study in wt mice; N=12.Robust and sustained Tau RNA (FIG. 9A) and protein (FIG. 9B) reductionwas achieved; 3×33 ug intra-cerebroventricular bolus injections producedsimilar results (data not shown).

Dose dependent Tau RNA reduction was also observed following intrathecal(IT) injection of oligomers similar to ASO-000013 and ASO-000757 intolumbar ported rats (data not shown). A single bolus IT injection of 300μg of ASO-000013 or ASO-000757 was injected into lumbar catheterizedrats (as described above). Robust and sustained reduction of brain TaumRNA was observed at both 3 days and 4 weeks following the single bolusadministration using the proposed clinical route of administration ofthese representative oligomers (FIG. 11). IT administration is thepreferred clinical route for the treatment of tau dependent disorders.

Tau Protein Reduction

Tau ASOs in the 3′UTR were administered at 100 μgintra-cerebroventricular (ICV) to hTau or wild type B16 mice in order tounderstand the hysteresis of Tau protein reduction with respect to mRNAreduction. During these studies, many of the Tau ASOs were not toleratedbeyond 4 weeks following a single 100 μg ICV bolus dose. Some of themost potent Tau ASOs in this region also reduced expression of anunintended target Ras homolog gene family, member A (“RhoA”). RhoA is asmall GTPase protein of Rho family. While the effects of RhoA activityare not all well known, it is primarily associated with cytoskeletonregulation, mostly actin stress fibers formation and actomyosincontractility. In humans, it is encoded by the gene RHOA. The RHOA genecontains the sequence of actttatttccaaatacacttcttt (SEQ ID NO: 959).FIG. 12 shows that the RHOA gene fragment has one to four basepairmismatches with selected oligomers (e.g., ASO-000757, ASO-000755, orASO-000753).

Certain traditional gapmer sequences were further modified in the gapdesign and the wing design. In particular, the traditional gapmer designwas converted to an alternating flank gapmer design (e.g., ASO-001967,ASO-001941, ASO-001933, and ASO-1940). FIG. 12 shows that thetraditional gapmers are not tolerated beyond 4 weeks following a single100 μg ICV bolus dose while the alternating flank gapmers exhibittolerability beyond 4 weeks.

FIG. 12 also shows that tubulin (Tuj 1) was highly correlated with longterm tolerability for the ASOs shown. Rho A reduction greater than 25%also correlated with lack of long term tolerability (greater than 4weeks following a single ICV bolus injection of 100 μg of each ASO shownin FIG. 12).

ASO-001933 (100 μg-200 μg) was administered as a single bolusintracerebroventricularly (ICV) in mice, as described above, andproduced greater than 50% reduction of brain Tau protein that wassustained for 4-12 weeks in hTau mice. At these dose levels, there wereno clinical signs of toxicity and no gross or histologic findingsobserved over the 20-week period following a single ICV dose in mouse.ASO-000013 was also administered and gave results similar to ASO-001933.A single ICV bolus injection of 100 μg produced no adverse changes incognition as assessed by novel object recognition or contextual fearconditioning, motor function as assessed by catwalk, rotorod and runningwheel (data not shown). In a Tau knock out mouse carrying the entirehuman tau gene (hTau), the EC₅₀ for reduction of human Tau brain mRNAand protein was ˜2.72 μg/g (414 nM). As FIG. 13 shows, ASO-001933 (TauASO) produces durable, dose responsive brain hTau protein reductionafter a single intracerebroventricular (ICV) injection in hTau mousebrain. Saline or 50,100, 150 and 200 μg of Tau ASO was injected ICV inhTau mice (n=10 per group). The frontal cortical region was dissectedeight weeks post dose to determine total Tau protein levels by ELISA(BT2/HT7). Two-way ANOVA and Bonferroni post hoc analysis were used***p<0.001. Error bars represent SEM.

The relationship between the level of brain Tau protein suppression andfunctional outcome measures was studied in both tauopathy (Tg4510) andDravet Syndrome mouse models. Initial data generated in these geneticmouse models (FIGS. 14 and 15) suggest that about 25-50% reduction ofbrain soluble and insoluble Tau protein compared to a control issufficient for potential functional improvement in tauopathies like PSPand/or intractable early childhood epilepsies like Dravet. Inparticular, FIG. 14A shows that a single 100 μg ICV bolus of ASO-000774reduced total Tau protein. The protein reduction was measured by usingBT-2 and HT-7 ELISA described herein. p<0.05 unpaired t-test. In FIG.14B, Tg4510 and double negative littermate controls (Dbl Neg) wereassessed in a running wheel assay as described above. A single 100 μgICV bolus of ASO-000774 reversed hyperactivity in Tg4510 to level of DblNeg littermate controls, p<0.05 Two-Way RMANOVA followed by Bonferroni'spost test.

In addition, about 25-50% reduction of brain soluble and insoluble Tauprotein compared to a control is sufficient to improve survival and heatinduced seizure in Dravet mice, which possess a mutation in the SCNA1gene (FIG. 15). Dravet mice treated with a single ICV administration of20 or 37 μg of Tau ASO-000762 targeting the 3′-UTR region of Tau mRNAexhibited about 20-50% Tau protein reduction (data not shown) at 10 dayspostnatally. In addition, as FIG. 15A shows, the Dravet mice showed agreater percentage of live mice between 30-55 days when compared withlittermate controls. Significant treatment effect has been shown by Coxproportional hazard regression. Dravet mice were tested to measurehyperthermia-induced Generalized Tonic-Clonic Seizures (GTCS). FIG. 15Bshows that ASO-000762 at 20 μg protected against hyperthermia-inducedGeneralized Tonic-Clonic Seizures (GTCS) in Dravet mice after 8-9 weekspost-injection. Consistent with the in vivo Dravet studies, ASO-001933tested in neurons derived from Dravet and human isogenic control Inducedpluripotent stem cells (“iPSCs”) corrected the network activity inducedby neurotransmitter(s) (data not shown).

Example 8 Construction of Oligomers Targeting 5′ UTR and/or Exon 2

A number of oligomers were designed to target the 5′ UTR and/or exon 2of MAPT pre-mRNA. See FIG. 1 for genomic MAPT sequence. For example, theoligomers were constructed to target nucleotides 72,802-73,072 of SEQ IDNO: 1. The exemplary sequences of the oligomers are described in FIGS.16A and B. In some embodiments, the oligomers were designed to begapmers or mixmers. FIGS. 16A and B show non-limiting examples of theoligomer design for selected sequences. The same methods can be appliedto any other sequences disclosed herein. The gapmers were constructed tocontain locked nucleic acids—LNAs (upper case letters). For example, agapmer can have Beta-deoxy LNA at the 5′ end and the 3′ end and have aphosphorothioate backbone. But the LNAs can also be substituted with anyother nucleotide analogs and the backbone can be other types ofbackbones (e.g., a phosphodiester linkage, a phosphotriester linkage, amethylphosphonate linkage, a phosphoramidate linkage, or combinationsthereof). A reference to a SEQ ID number includes a particular sequence,but does not include an oligomer design.

The oligomers were synthesized using methods well known in the art.Exemplary methods of preparing such oligomers are described inBarciszewski et al., Chapter 10—“Locked Nucleic Acid Aptamers” inNucleic Acid and Peptide Aptamers: Methods and Protocols, vol. 535,Gunter Mayer (ed.) (2009), the entire contents of which is herebyexpressly incorporated by reference herein.

Example 9 Tau mRNA and Protein Reduction in Cynomolgus Monkeys

Progressive supranuclear palsy (PSP) is a neurodegenerative syndromethat is clinically characterized by progressive postural instability,supranuclear gaze palsy, parkinsonism and cognitive impairment. PSP isdefined neuropathologically by the accumulation of tau-positiveneurofibrillary tangles in brain regions extending from the cerebralcortex, basal ganglia to the cerebellum and brainstem. The most severelyaffected brain regions include the brainstem substantia nigra, pontinenuclei and the cerebellar dentate nucleus. Tauopathy in these regions isbelieved to underpin several clinical features of PSP such as posturalinstability, dysarthria and gaze palsy. Suppression of Tau mRNAtranscripts and, consequently, protein in the brain regions, can havetherapeutic significance for treatment of PSP patients.

Subjects were male cynomolgus monkeys weighing 3.5-10.0 kg at the startof the study. Each was implanted with an intrathecal CSF catheterentering at the L3 or L4 vertebrae extending to approximately the L1vertebra. The proximal end of the catheter was connected to asubcutaneous access port. CSF was collected through the port by gravityflow to a maximum of 0.5 ml CSF per sample. The CSF was centrifuged andthe supernatent was kept at −90° C. until analyzed. Blood plasmaobtained from an available vein was kept at −90° C. until analyzed.

Cynomolgus monkeys were administered with ASO-1933, which was dissolvedin saline, at 0.33 ml/min in a 1.0 ml volume followed by a 0.5 mlsterile water flush. Total infusion time was 4.5 min.

Cynomolgus monkeys were administered the appropriate volume of acommercially available euthanasia solution while anesthetized withketamine and isoflurane. Necropsy tissues were obtained immediatelythereafter and the brain was transferred to wet ice for dissection.Areas of interest were dissected using 6 mm slices in an ASI Cyno BrainMatrix as well as free handed techniques. Samples were placed fresh inRNAlater, or frozen on dry ice for later analysis. Some slices werefrozen intact for immunohistochemical analysis. Slices were placed in aweigh boat and floated on isopentane cooled with dry ice. Once frozen,slices were stored at −90° C. until analysis.

For brain block sectioning, the frozen brain blocks were cut on acryostat coronal sections, and sections were thaw-mounted onto superfrost slides, dried, re-frozen on dry ice, and stored at −80° C. untiluse. Brain sections collected from the cynomolgus monkey dosed withvehicle, ASO-1933 at 16 mg (1×16) or ASO-1933 at 16 mg twice (2×16, with2 weeks apart) were used for the in situ hybridization (ISH) study.

In order to measure Tau mRNA expression using [³⁵S]labeled antisenseISH, a Tau DNA template and [³⁵S]labeled antisense probes weresynthesized. A Tau DNA template (425 bp, 687-1111, accession number:XM_005584540.1) was amplified from a cynomolgus monkey cDNA library(Zyagen KD-201) by PCR using forward primer 5′-CAA GCT CGC ATG GTC AGTAA-3′ (SEQ ID NO: 954) and reverse primer 5′-AAT TAA CCC TCA CTA AAG GGAGA TTC TCA GTG GAG CCG ATC TT-3′ (SEQ ID NO: 955). Products of desiredsize were observed by gel electrophoresis. The Tau DNA template wastranscripted with T3 RNA polymerase (Invitrogen AM1316) using [³⁵S]UTP(Perkin Elmer NEG-739) to produce a [³⁵S]labeled antisense ISH probe.

To measure Tau mRNA ISH using [³⁵S]labeled antisense probe, slides werethawed, fixed in 4% paraformaldehyde for 15 min at 4° C. followed byrinsing. Slides were then treated in acetic anhydride/triethanolaminefollowed by rinsing. Slides were pre-hybridized in pre-hybridizationsolution at 50° C. for 3 hours and hybridized with 1.5×10⁴ cpm/ul[³⁵S]riboprobe (0.75 ml/slide) in hybridization solution. Afterhybridization, slides were washed at room temperature. Slides were thentreated with Rnase A at 37° C., washed twice, followed by a highstringency wash. The sections were dehydrated in 90% alcohol containing0.3 M NH₄Ac, dried, and exposed against phosphor screen (Perkin ElmerPPN 7001487). After exposure, autoradiographic images on the screen werecaptured and analyzed using Cyclone storage phosphor system andOptiQuant Acquisition and Analysis software (PerkinElmer, Waltham,Mass.).

QuantiGene® ViewRNA tissue ISH was used to detect Tau mRNA expression atthe subnucleus and cellular levels. An antisense probe (type-1)targeting Tau mRNA (2344-3300, accession number: XM_005584529) wassynthesized by Affymetrix. Slides were fixed in 4% formaldehyde inphosphate buffered saline (PBS). After passing through alcohol gradientsfor 10 minutes each, slides were dried, followed by protease QFdigestion. Subsequently, sections were washed and hybridized with thetarget probe. Slides were then washed in wash buffer and stored instorage buffer overnight. Slides were then processed through a series ofsequential PreAmp and Amp hybridization steps. The sections wereincubated with Label Probe AP followed by incubation with Fast RedSubstrate, rinsed in PBS, and counterstained using either Gill'sHematoxylin or DAPI. Slides were coverslipped using DAKO ultramountmounting medium and stored. Labeled Tau mRNA was visualized using eithera Leica brightfield microscope or a Leica confocal fluorescencemicroscope (excitation: 630 nm; emission: 760).

To measure Tau protein expression, Tau12 (BioLegend, San Diego, Calif.,epitope to amino acids 6-18 on tau 441 sequence) and BT2 (ThermoScientific, Rockville, Ill., epitope to amino acids 194-198) were usedto coat Costar 3925 ELISA plates at 2.5 and 1 μg/ml, respectively.Plates were incubated for 1 h at 37° C. before washing with TBS with0.05% Tween-20 (TBST). Non-specific binding was blocked by the additionof 3% bovine serum albumin (BSA) in TBS with 0.1% Tween-20 for 4 h atroom temperature with shaking. Plates were washed with TBST before theaddition of samples or standard curve generated with recombinanth-tau441 protein, both of which were prepared in TBST plus 1% BSA.Plates containing standard curve and samples were incubated overnight at4° C. with shaking. The following detection antibodies were conjugatedwith alkaline phosphatase (AP) using the Lightning Link Conjugation Kit(Novus Biologicals, Littleton, Colo.): BT2 and HT7 (Thermo Scientific,epitope of 159-163). AP-conjugated detection antibodies were diluted inTBST plus 1% BSA and co-incubated with samples and standard curve for 1h at room temperature with shaking. After washing with TBST, TropixCDP-Star Ready-to-Use with Sapphire-II AP substrate (Applied Biosystems,Bedford, Mass.) was added for 30 min. Chemiluminescent signal wasdetermined using a Perkin Elmer EnVision microplate reader (Waltham,Mass.).

The N-terminal tau sandwich ELISA (Tau12-BT2) consists of the anti-tauantibody Tau12 as capture and detection with an alkaline phosphatase(AP) conjugate of the anti-tau antibody BT2. The mid-domain tau sandwichELISA (BT2-HT7) consists of the anti-tau antibody BT2 as the captureantibody and detection with an alkaline phosphatase (AP) conjugate ofthe anti-tau antibody HT7.High binding black well ELISA plates (Costar,Corning, Tewksbury, Mass.) were coated with anti-Tau BT2 monoclonalantibody (Thermo, Waltham, Mass.) at 2.5 μg/ml or Tau12 anti-taumonoclonal antibody (Covance) at 5 μg/ml in tris buffered saline (50μL/well). The plates were washed with tris buffered saline containing0.05% tween-20 (TBS-T) followed by blocking at room temperature withshaking in 3% BSA/TBS (BSA from Roche, Indianapolis, Ind.). The plateswere rewashed as listed above followed by sample addition in triplicate(50 μL/well). Cynomolgus monkey CSF samples were diluted 1:30 (BT2/HT7)or 1:25 (Tau12/BT2) in 1% BSA/TBS-T. A Tau 441 (R-peptide, Bogart, Ga.)standard curve was made. The samples were incubated on the ELISA plateovernight at 4° C. with shaking. AP conjugated HT7 or BT2 was diluted to0.25 μg/ml (HT7) or 0.1 μg/ml (BT2) in 1% BSA/TBS-T was added to theplates (50 μL/well) for co-incubation with standards and samples for 1hour at room temperature with shaking. The plates were re washedfollowed by the addition of chemiluminescent substrate (Tropix CDP Star,Applied Biosystems, Grand Island, N.Y.) (100 μL/well) and incubation atroom temperature with shaking for 30 minutes. The plates were read on aPerkin Elmer TopCount. Unknown sample values were read off the Tau-441standard curve using GraphPad Prism software.

These studies demonstrate that intrathecally-applied Tau ASO distributesto the substantia nigra, pontine nuclei and dentate nucleus andsuppresses Tau mRNA expression in these brain regions in Cynomolgusmonkeys following intrathecal administration of ASO-001933 following twodoses (2 week apart) of 16 mg (2×16). FIG. 17A show in situhybridization (ISH) autoradiographic images of tau mRNA expression(lighter shades) in the substantia nigra, pontine nuclei and dentatenucleus in the monkeys dosed with vehicle or ASO-001933 2×16 mg (1 weekapart). As FIG. 17B shows, ASO-001933 produced profound suppression ofTau mRNA expression in all three regions in both monkeys. The Tau mRNAknockdown effect produced by ASO-001933 was further demonstrated usingthe QuantiGene® ViewRNA ISH assay (data not shown). FIG. 17B shows thatin the vehicle-treated monkey, a high intensity Tau mRNA labeling waspresent, primarily, in neuronal cell bodies in the substantia nigra,pontine nuclei and dentate nucleus. In cynomolgus monkeys, two single 16mg intrathecal doses of ASO-001933, one week apart were administered toassess anatomic distribution of Tau mRNA reduction in anatomic brainregions where pathologic Tau accumulates in PSP (FIG. 17A).

In monkeys, a single intrathecal (IT) dose of 4 mg of ASO-001933produced Tau mRNA reductions between 58% to 80% in cortical brainregions and 63% in cerebellum within 2 weeks post dose (data not shown).These areas of the brain are believed to be important for treatment ofTau-dependent dysfunction in PSP (neurodegenerative tauopathies) andDravet syndrome (epilepsy and autism spectrum disorders), leadingindications for Tau antisense molecules like ASO-001933.

Consistent with Tau mRNA, FIGS. 18A and 18B shows that the ASO-001933administration as IT bolus injection (2 doses of 8 mg given 2 weeksapart) in monkeys is capable of reducing about 70% of Tau protein in thebrain (FIG. 18A) about 60% in the CSF (FIG. 18B). The Tau proteinexpression was observed 12 weeks following the ASO administration.Similarly, the ASO-001933 administration as a single ICVintra-cerebroventricular injection (100 μg) in mice is capable ofreducing about 50% of Tau protein in the brain (data not shown) andabout 34% of Tau protein in the CSF (data not shown). These data suggestthat reduction of CSF Tau protein can be a clinically accessiblebiomarker of target engagement.

ASO-002038 was administered as a single bolus intracerebroventricularly(FIG. 19A: ICV at 25-150 μg) or intrathecally (FIG. 19B: IT at 400-900μg) in mice or in rats, as described in Example 5. ASO-002038 produceddose dependent hTau mRNA reduction in the brain with a calculated EC₅₀value ˜598 nM in mice. At these dose levels, there were no clinicalsigns of toxicity and no gross or histologic findings observed followinga single ICV dose in mouse. Many ASOs including ASO-000013, ASO-001933,ASO-001967, ASO-001940, ASO-001941, and others produced similar hTaudose dependent reduction, EC₅₀ for reduction of human Tau brain mRNA,and were well tolerated in mice (data not shown).

Example 10 Quantigene Analysis of Tau, Rho and Tubulin mRNA Expression

To measure tau, rhoA and tubulin mRNA reduction, primary neuronalcultures were established from the forebrain of E18 transgenic miceexpressing the human tau transgene on a mouse tau knockout background.(Andorfer et al. J Neurochem 86:582-590 (2003)). Cultures were preparedas described in Example 2. Alternatively, iNeurons from CellularDynamics Inc., were used per manufacturer specifications.

Lysis: Cells were plated on poly-D-lysine coated 96 well plates at50,000 cells per well and maintained in Neurobasal media containing B27,glutamax and Penicillin-Streptomycin. ASOs were diluted in water andadded to cells at DIV01 to a final concentration of 5 □M. For IC₅₀determinations, neurons were treated with a top concentration of 5 uMand a concentration response dilution of 1:3 was used to define theIC₅₀. Following ASO treatment, neurons were incubated at 37° C. for 5days to achieve steady state reduction of mRNA. Media was removed andcells were washed 1× in DPBS and lysed as follows. Measurement of lysatemessenger RNA was performed using the Quantigene 2.0 Reagent System(Affymetrix), which quantitates RNA using a branched DNA-signalamplification method reliant on the specifically designed RNA captureprobe set. The working cell lysis buffer solution was made by adding 50μl proteinase K to 5 ml of pre-warmed Lysis mix and diluted to 1:4 finaldilution with dH₂O. The working lysis buffer was added to the plate (150μl/well), triturated to mix, sealed and incubated. Following lysis thewells were titrated to mix and stored at −80° C. or assayed immediately.

Assay: Lysates were diluted in lysis mix dependent on the specificcapture probe used (tau,RhoA or tubulin). 80 μl/well total were thenadded to the capture plate (96 well polystyrene plate coated withcapture probes). Working probe sets reagents were generated by combiningnuclease-free water 12.1 μl, lysis mixture 6.6 μl, blocking reagent 1specific 2.0 probe set 0.3 μl (human MAPT catalogue #15486, human RHOAcatalogue #SA-11696, or human beta 3 tubulin catalogue #SA-15628) permanufacturer instructions (QuantiGene 2.0 Affymetrix). Then 20 μlworking probe set reagents were added to 80 μl lysate dilution (or 80 μllysis mix for background samples) on the capture plate. Plates werecentrifuged and then incubated for 16-20 hours at 55° C. to hybridize(target RNA capture). Signal amplification and detection of target RNAwas begun by washing plates with buffer 3 times to remove unboundmaterial. 2.0 Pre-Amplifier hybridization reagent (100 μl/well) wasadded, incubated at 55° C. for 1 hour then aspirated and wash buffer wasadded and aspirated 3 times. The 2.0 Amplifier hybridization reagent wasthen added as described (100 μl/well), incubated for 1 hour at 55° C.and the wash was repeated as described previously. The 2.0 Label Probehybridization reagent was added next (100 μl/well), incubated for 1 hourat 50° C. and the wash was repeated as described previously. Lastly, theplates were centrifuged to remove any excess wash buffer and 2.0Substrate was added (100 μl/well). Plates were incubated for 5 minutesat room temperature and plates were imaged on a PerkinElmer Envisionmultilabel reader in luminometer mode within 15 minutes.

Data determination: For the gene of interest, the average assaybackground signal was subtracted from the average signal of eachtechnical replicate. The background-subtracted, average signals weredivided by the background subtracted average signal for the housekeepingtubulin RNA. The percent inhibition for the treated sample wascalculated relative to control treated sample lysate. Results ofQuantigene assays for cells treated with the oligomers (ASOs) are shownin FIGS. 20A and 20B.

The invention claimed is:
 1. An oligomer consisting of the nucleic acidsequence ATTtCcaaattcacTtTtAC (SEQ ID NO:487) or ATtTCcaaattcactTTtAC(SEQ ID NO:472), wherein each upper case letter is a beta-D-oxy-LNAnucleoside, and wherein each lower case letter is a DNA nucleoside.
 2. Aconjugate comprising the oligomer of claim 1, wherein the oligomer iscovalently attached to at least one non-nucleotide or non-polynucleotidemoiety.
 3. A pharmaceutical composition comprising the oligomer of claim1 and a pharmaceutically acceptable diluent, carrier, salt, or adjuvant.4. The pharmaceutical composition of claim 3, which is capable ofdown-regulating expression of the MAPT mRNA in a human cell.
 5. Thepharmaceutical composition of claim 4, wherein the human cell is aneuronal cell.
 6. A pharmaceutical composition comprising the conjugateof claim 2 and a pharmaceutically acceptable diluent, carrier, salt, oradjuvant.
 7. A method of inhibiting or reducing Tau protein expressionin a cell, the method comprising: administering a pharmaceuticalcomposition comprising at least one of: an oligomer consisting of thenucleic acid sequence ATTtCcaaattcacTtTtAC (SEQ ID NO:487) orATtTCcaaattcactTTtAC (SEQ ID NO:472), wherein each upper case letter isa beta-D-oxy-LNA nucleoside, and wherein each lower case letter is a DNAnucleoside; or a conjugate comprising an oligomer having a nucleic acidsequence comprising ATTtCcaaattcacTtTtAC (SEQ ID NO:487) orATtTCcaaattcactTTtAC (SEQ ID NO:472), wherein each upper case letter isbeta-D-oxy-LNA nucleoside, and wherein each lower case letter is a DNAnucleoside, wherein the oligomer is covalently attached to at least onenon-nucleotide or non-polynucleotide moiety; to the cell expressing Tauprotein, wherein the Tau protein expression in the cell is inhibited orreduced after the administration.
 8. The method of claim 7, wherein thepharmaceutical composition further comprises a pharmaceuticallyacceptable diluent, carrier, salt, or adjuvant.
 9. A method for treatinga tauopathy, the method comprising: administering an effective amount ofa pharmaceutical composition comprising at least one of: an oligomerconsisting of the nucleic acid sequence ATTtCcaaattcacTtTtAC (SEQ IDNO:487) or ATtTCcaaattcactTTtAC (SEQ ID NO:472), wherein each upper caseletter is a beta-D-oxy-LNA nucleoside, and wherein each lower caseletter is a DNA nucleoside; or a conjugate comprising an oligomerconsisting of the nucleic acid sequence ATTtCcaaattcacTtTtAC (SEQ IDNO:487) or ATtTCcaaattcactTTtAC (SEQ ID NO:472), wherein each upper caseletter is a beta-D-oxy-LNA nucleoside, and wherein each lower caseletter is a DNA nucleoside, wherein the oligomer is covalently attachedto at least one non-nucleotide or non-polynucleotide moiety.
 10. Themethod of claim 9, wherein the pharmaceutical composition furthercomprises a pharmaceutically acceptable diluent, carrier, salt, oradjuvant.
 11. A method for treating a disorder associated with overexpression or expression of a mutated version of Tau protein, the methodcomprising: administering an effective amount of a pharmaceuticalcomposition comprising at least one of: an oligomer consisting of thenucleic acid sequence ATTtCcaaattcacTtTtAC (SEQ ID NO:487) orATtTCcaaattcactTTtAC (SEQ ID NO:472), wherein each upper case letter isa beta-D-oxy-LNA nucleoside, and wherein each lower case letter is a DNAnucleoside; or a conjugate comprising an oligomer consisting of thenucleic acid sequence ATTtCcaaattcacTtTtAC (SEQ ID NO:487) orATtTCcaaattcactTTtAC (SEQ ID NO:472), wherein each upper case letter isa beta-D-oxy-LNA nucleoside, and wherein each lower case letter is a DNAnucleoside, wherein the oligomer is covalently attached to at least onenon-nucleotide or non-polynucleotide moiety.
 12. The method of claim 11,wherein the pharmaceutical composition further comprises apharmaceutically acceptable diluent, carrier, salt, or adjuvant.
 13. Themethod of claim 11, wherein the disorder associated with over expressionor expression of a mutated version of Tau protein is a tauopathy.